Enhanced pyruvate to 2,3-butanediol conversion in lactic acid bacteria

Abstract

A high flux of metabolites from pyruvate to 2,3-butanediol in Lactobacillus plantarum was achieved through genetic engineering. Substantial elimination of lactate dehydrogenase activity in the presence of heterologously expressed butanediol dehydrogenase activity led to 2,3 butanediol production that was at least 49% of the total of major pyruvate-derived products.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is related to and claims the benefit of priority to U.S. Provisional Application No. 61/100,786, filed Sep. 29, 2008, the entirety of which is herein incorporated by reference.

Description

FIELD OF THE INVENTION

The invention relates to the field of industrial microbiology and the metabolism of lactic acid bacteria. More specifically, engineering lactic acid bacteria for a high flux from pyruvate to 2,3-butanediol allows increased production of 2,3-butanediol and compounds in pathways including 2,3-butanediol as an upstream substrate.

BACKGROUND OF THE INVENTION

2,3-butanediol, 2-butanone, and 2-butanol are important industrial chemicals. 2,3-butanediol may be used in the chemical synthesis of butene and butadiene, important industrial chemicals currently obtained from cracked petroleum, and esters of 2,3-butanediol may be used as plasticizers (Voloch et al. Fermentation Derived 2,3-Butanediol, in Comprehensive Biotechnology, Pergamon Press Ltd, England Vol 2, Section 3:933-947 (1986)). 2-Butanone, also referred to as methyl ethyl ketone (MEK), is a widely used solvent and is the most important commercially produced ketone, after acetone. It is used as a solvent for paints, resins, and adhesives, as well as a selective extractant, activator of oxidative reactions, and it can be chemically converted to 2-butanol by reacting with hydrogen in the presence of a catalyst (Nystrom, R. F. and Brown, W. G. (J. Am. Chem. Soc. (1947) 69:1198). Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase.

Microorganisms may be engineered for expression of biosynthetic pathways for production of 2,3-butanediol, 2-butanone, and/or 2-butanol. US Patent Pub US20070292927A1 discloses the engineering of recombinant microorganisms for expression of a biosynthetic pathway having 2,3-butanediol and 2-butanone as intermediates and 2-butanol as the end product. The pathway initiates with cellular pyruvate. Thus production of 2,3-butanediol, 2-butanone, and 2-butanol is limited by the availability of pyruvate substrate flow from natural host pathways into this engineered biosynthetic pathway.

In lactic acid bacteria, a limited amount of 2,3-butanediol may be produced naturally, but the major pyruvate metabolic pathway is conversion to lactate through activity of lactate dehydrogenase (LDH). Metabolic engineering to redirect pyruvate from lactate to other products in lactic acid bacteria has had unpredictable results. Production of alanine in LDH-deficient Lactococcus lactis expressing alanine dehydrogenase was shown by Hols et al. (Nature Biotech. 17:588-592 (1999). However, production of ethanol in LDH-deficient Lactobacillus plantarum expressing pyruvate decarboxylase was very limited, with carbon flow not significantly improved toward ethanol and lactate still produced (Liu et al. (2006) J. Ind. Micro. Biotech. 33:1-7).

Where a lactic acid bacteria is the preferred host for the production of 2-butanol and 2-butanone, a need exists therefore for lactic acid bacteria to have a tightly regulated carbon flow from pyruvate to 2,3-butanediol. To date no bacteria has been engineered to produce this advantage and the art suggests that simply reducing the carbon flow from pyruvate to lactate via lactate dehydrogenase may not be sufficient. Applicants have solved the stated problem through the unexpected discovery that introduction of a heterologous polypeptide having butanediol dehydrogenase activity in combination with reduction in endogenous lactate dehydrogenase results in unpredictably high rates of conversion of pyruvate to down stream products and particularly 2,3-butanediol.

SUMMARY OF THE INVENTION

Provided herein are recombinant lactic acid bacterial cells comprising at least one gene encoding a heterologous polypeptide having butanediol dehydrogenase activity wherein the bacterial cell is substantially free of lactate dehydrogenase activity and wherein the cell produces 2,3-butanediol. In one embodiment, the bacterial cell comprises a disruption in at least one endogenous gene encoding a polypeptide having lactate dehydrogenase activity. In one embodiment, the cell is a member of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

In one embodiment, the cell comprises at least one genetic modification that reduces pyruvate formate lyase activity. In some embodiments, the genetic modification affects a gene encoding pyruvate formate lyase, a gene encoding pyruvate formate lyase activating enzyme, or both. In some embodiments, the gene encoding pyruvate formate lyase is selected from the group consisting of pfl, pflB1 and pfl B2 and the gene encoding formate C-acetyltransferase activating enzyme is selected from the group consisting of pflA, pflA1, and pflA2.

Also provided are embodiments wherein the cell produces a product selected from the group consisting of lactate, acetoin, ethanol, succinate, and formate. In some embodiments, 2,3-butanediol comprises at least about 49 Mol % of all products produced from pyruvate.

In some embodiments, the polypeptide having lactate dehydrogenase activity is encoded by a gene selected from the group consisting of ldhL, ldhD, ldhL1, and ldhL2.

In one embodiment, the lactic acid host cell is Lactobacillus plantarum and the polypeptide having lactate dehydrogenase activity has an amino acid sequence that has at least about 95% identity to a sequence selected from the group consisting of SEQ ID NO: 2,4, and 6. In one embodiment, the lactic acid host cell is Lactococcus lactis and the polypeptide having lactate dehydrogenase activity has an amino acid sequence that has at least about 95% identity to the sequence as set forth in SEQ ID NO:20. In another embodiment, lactic acid host cell is Leuconostoc mesenteroides and the polypeptide having lactate dehydrogenase activity has an amino acid sequence that has at least about 95% identity to the sequence as set forth in SEQ ID NO:22. In another embodiment, the lactic acid host cell is Streptococcus thermophilus and the polypeptide having lactate dehydrogenase activity has an amino acid sequence that has at least about 95% identity to the sequence as set forth in SEQ ID NO:24. In another embodiment, the lactic acid host cell is Pediococcus pentosaceus and the polypeptide having lactate dehydrogenase activity has an amino acid sequence that has at least about 95% identity to a sequence selected from the group consisting of SEQ ID NO:26 and 28. In another embodiment, the lactic acid host cell is Lactobacillus acidophilus and the polypeptide having lactate dehydrogenase activity has an amino acid sequence that has at least about 95% identity to a sequence selected from the group consisting of SEQ ID NO:30, 32 and 34.

In one embodiment, the heterologous polypeptide having butanediol dehydrogenase activity has an amino acid sequence that has at least about 95% identity to a sequence selected from the group consisting of SEQ ID NO: 13, 64 and 66.

In one embodiment, the cell produces 2-butanone, and in one embodiment, the cell comprises a 2-butanone biosynthetic pathway. In one embodiment, the cell produces 2-butanol, and in one embodiment, the cell produces a 2-butanol biosynthetic pathway.

Also provided herein are methods for the production of 2-butanol comprising: providing a recombinant lactic acid bacterial cell comprising a 2-butanol biosynthetic pathway; and growing the bacterial cell of step (a) under conditions whereby 2-butanol is produced.

Also provided are methods for the production of 2-butanone comprising: providing a recombinant lactic acid bacterial cell comprising a 2-butanone biosynthetic pathway; and b) growing the bacterial cell of step (a) under conditions whereby 2-butanone is produced.

BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS

The various embodiments of the invention can be more fully understood from the following detailed description, the figures, and the accompanying sequence descriptions, which form a part of this application.

FIG. 1 shows a biosynthetic pathway for biosynthesis of 2,3-butanediol, 2-butanone, and 2-butanol.

FIG. 2 shows a graph of products made in L. plantarum strains PN0512 (control) and PNP0001 (ldhDldhL1 deletion strain).

FIG. 3 shows a graph of products made in L. plantarum strains BP134 (control with budC and sadB genes), PNP0001 (ldh deletion), and PNP0002 (ldh deletion with budC and sadB genes) grown in rich medium.

FIG. 4 illustrates common lactate fermentation pathways in lactic acid bacteria.

The invention can be more fully understood from the following detailed description and the accompanying sequence descriptions which form a part of this application.

The following sequences conform with 37 C.F.R. 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures--the Sequence Rules") and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. .sctn.1.822.

TABLE-US-00001 TABLE 1 SEQ ID NOs of lactate dehydrogenase coding regions and proteins SEQ ID NO: SEQ ID NO: Organism and gene name nucleic acid amino acid Lactobacillus plantarum ldhD 1 2 Lactobacillus plantarum ldhL1 3 4 Lactobacillus plantarum ldhL2 5 6 Lactococcus lactis ldhL 19 20 Leuconostoc mesenteroides ldhD 21 22 Streptococcus thermophilus ldhL 23 24 Pediococcus pentosaceus ldhD 25 26 Pediococcus pentosaceus ldhL 27 28 Lactobacillus acidophilus ldhL1 29 30 Lactobacillus acidophilus ldhL2 31 32 Lactobacillus acidophilus ldhD 33 34

TABLE-US-00002 TABLE 2 SEQ ID NOs of butanediol dehydrogenase coding regions and proteins SEQ ID NO: SEQ ID NO: Description nucleic acid amino acid budC, butanediol dehydrogenase from 12 13 Klebsiella pneumoniae IAM1063 butanediol dehydrogenase from 63 64 Bacillus cereus butB, butanediol dehydrogenase from 65 66 Lactococcus lactis

TABLE-US-00003 TABLE 3 SEQ ID NOs of pyruvate formate lyase and pyruvate formate lyase activating enzyme coding regions and proteins SEQ ID NO: SEQ ID NO: Organism and gene name nucleic acid amino acid PflB1 from Lactobacillus plantarum 69 70 PflB2 from Lactobacillus plantarum 71 72 PflA1 from Lactobacillus plantarum 73 74 PflA2 from Lactobacillus plantarum 75 76 Pfl from Lactococcus lactis 77 78 PflA from Lactococcus lactis 79 80 Pfl from Streptococcus thermophilus 81 82 PflA from Streptococcus thermophilus 83 84

TABLE-US-00004 TABLE 4 SEQ ID NOs of expression coding regions and proteins SEQ ID NO: SEQ ID NO: Description nucleic acid amino acid Achromobacter xylosoxidans secondary 9 10 alcohol dehydrogenase sadB Roseburia inulinivorans butanediol 15 16 dehydratase rdhtA Roseburia inulinivorans butanediol 17 18 dehydratase reactivase rdhtB ALS from Bacillus subtilis 85 86 ALS from Bacillus subtilis coding region 87 86* optimized for Lactobacillus plantarum ALS from Klebsiella pneumoniae (budB) 88 89 ALS from Lactococcus lactis 90 91 ALS from Staphylococcus aureus 92 93 ALS from Listeria monocytogenes 94 95 ALS from Streptococcus mutans 96 97 ALS from Streptococcus thermophilus 98 99 ALS from Vibrio angustum 100 101 ALS from Bacillus cereus 102 103 *same protein sequence encoded by native and optimized sequence

SEQ ID NO:7 is the nucleotide sequence of the coding region for orotidine-5'-phosphate decarboxylase from L. plantarum.

SEQ ID NO:8 is the nucleotide sequence of the L. plantarum ldhL1 promoter.

SEQ ID NO:11 is the nucleotide sequence of the S. cerevisiae FBA promoter.

SEQ ID NO:14 is the nucleotide sequence of the S. cerevisiae GPM1 promoter.

SEQ ID NOs:35-38 are plasmids pFP996, pFP996PldhL1, pFP996PldhL1-budC-sadB, and pFP996PldhL1-budC, respectively.

SEQ ID NOs:39-50, 52-62, and 104-113 are PCR, sequencing or cloning primers.

SEQ ID NO:51 is the nucleotide sequence of a ribosome binding site.

SEQ ID NO:67 is the sequence of a synthetic fragment containing coding regions for Roseburia inulinivorans B.sub.12-independent diol dehydratase and reactivase.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to recombinant lactic acid bacterial (LAB) cells that are genetically modified to have improved conversion of pyruvate, and in particular endogenous pyruvate, to 2,3-butanediol. The LAB cells express a heterologous butanediol dehydratase and are substantially free of lactate dehydrogenase activity. In addition, the present invention relates to methods of producing 2,3-butanediol, 2-butanone, or 2-butanol using the present genetically modified LAB cells. Production of these compounds in lactic acid bacteria will reduce the need for petrochemicals for their production as industrial chemicals for applications as solvents and/or extractants, and these compounds may replace fossil fuels either directly or as intermediates for further chemical synthesis of fossil fuel replacements.

The following abbreviations and definitions will be used for the interpretation of the specification and the claims.

As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains" or "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, a mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances (i.e. occurrences) of the element or component. Therefore "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

The term "invention" or "present invention" as used herein is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the specification and the claims.

As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or use solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about", the claims include equivalents to the quantities. In one embodiment, the term "about" means within 10% of the reported numerical value, preferably within 5% of the reported numerical value.

The term "2-butanol biosynthetic pathway" refers to an enzyme pathway to produce 2-butanol from pyruvate.

The term "2-butanone biosynthetic pathway" refers to an enzyme pathway to produce 2-butanone from pyruvate

The term "butanediol dehydrogenase" also known as "acetoin reductase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanediol dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)-stereochemistry in the alcohol product. (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (DNA: SEQ ID NO: 12, protein: SEQ ID NO: 13). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (DNA: SEQ ID NO:63, protein: SEQ ID NO:64), and Lactococcus lactis (DNA: SEQ ID NO:65, protein: SEQ ID NO:66).

The term "lactate dehydrogenase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of pyruvate to lactate. Lactate dehydrogenases are known as EC 1.1.1.27 (L-lactate dehydrogenase) or EC 1.1.1.28 (D-lactate dehydrogenase) and are further described herein.

The term "substantially free" when used in reference to the presence or absence of lactate dehydrogenase enzyme activity means that the level of the enzyme is substantially less than that of the same enzyme in the wild-type host, where less than 50% of the wild-type level is preferred and less than about 90% of the wild-type level is most preferred. The reduced level of enzyme activity may be attributable to genetic modification genes encoding this enzyme such that expression levels of the enzyme are reduced.

The term "a facultative anaerobe" refers to a microorganism that can grow in both aerobic and anaerobic environments.

The term "carbon substrate" or "fermentable carbon substrate" refers to a carbon source capable of being metabolized by host organisms of the present invention and particularly carbon sources selected from the group consisting of monosaccharides, oligosaccharides, polysaccharides, and one-carbon substrates or mixtures thereof.

The term "additional electron sink" refers to an electron sink or production of an electron sink that is not included in the biosynthetic pathway for the desired product.

The term "gene" refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. "Native gene" refers to a gene as found in nature with its own regulatory sequences. "Chimeric gene" refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. "Endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign gene" or "heterologous gene" refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. "Heterologous gene" includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene. For example, a heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Also a foreign gene can comprise native genes inserted into a non-native organism, or chimeric genes. A "transgene" is a gene that has been introduced into the genome by a transformation procedure.

As used herein the term "coding region" refers to a DNA sequence that codes for a specific amino acid sequence. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structure.

The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term "expression", as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide.

As used herein the term "transformation" refers to the transfer of a nucleic acid fragment into a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

The terms "plasmid" and "vector" as used herein, refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

As used herein the term "codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

The term "codon-optimized" as it refers to genes or coding regions of nucleic acid molecules for transformation of various hosts, refers to the alteration of codons in the gene or coding regions of the nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.

As used herein, an "isolated nucleic acid fragment" or "isolated nucleic acid molecule" will be used interchangeably and will mean a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid fragment is "hybridizable" to another nucleic acid fragment, such as a cDNA, genomic DNA, or RNA molecule, when a single-stranded form of the nucleic acid fragment can anneal to the other nucleic acid fragment under the appropriate conditions of temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989), particularly Chapter 11 and Table 11.1 therein (entirely incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. Stringency conditions can be adjusted to screen for moderately similar fragments (such as homologous sequences from distantly related organisms), to highly similar fragments (such as genes that duplicate functional enzymes from closely related organisms). Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2.times.SSC, 0.5% SDS was increased to 60.degree. C. Another preferred set of highly stringent conditions uses two final washes in 0.1.times.SSC, 0.1% SDS at 65.degree. C. An additional set of stringent conditions include hybridization at 0.1.times.SSC, 0.1% SDS, 65.degree. C. and washes with 2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible. The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of Tm for hybrids of nucleic acids having those sequences. The relative stability (corresponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations with shorter nucleic acids, i.e., oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al., supra, 11.7-11.8). In one embodiment the length for a hybridizable nucleic acid is at least about 10 nucleotides. Preferably a minimum length for a hybridizable nucleic acid is at least about 15 nucleotides; more preferably at least about 20 nucleotides; and most preferably the length is at least about 30 nucleotides. Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the probe.

A "substantial portion" of an amino acid or nucleotide sequence is that portion comprising enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to putatively identify that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Altschul, S. F., et al., J. Mol. Biol., 215:403-410 (1993)). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a "substantial portion" of a nucleotide sequence comprises enough of the sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence. The instant specification teaches the complete amino acid and nucleotide sequence encoding particular proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to one another. For example, with respect to DNA, adenosine is complementary to thymine and cytosine is complementary to guanine.

The term "percent identity", as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: 1.) Computational Molecular Biology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.) Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY (1991).

Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences is performed using the "Clustal method of alignment" which encompasses several varieties of the algorithm including the "Clustal V method of alignment" corresponding to the alignment method labeled Clustal V (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci., 8:189-191 (1992)) and found in the MegAlign.TM. program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). For multiple alignments, the default values correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences using the Clustal V program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program. Additionally the "Clustal W method of alignment" is available and corresponds to the alignment method labeled Clustal W (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992), Thompson, J. D., Higgins, D. G., and Gibson T. J. (1994) Nuc. Acid Res. 22: 4673 4680) and found in the MegAlign.TM. v6.1 program of the LASERGENE bioinformatics computing suite (DNASTAR Inc.). Default parameters for multiple alignment (GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergen Seqs(%)=30, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB). After alignment of the sequences using the Clustal W program, it is possible to obtain a "percent identity" by viewing the "sequence distances" table in the same program.

It is well understood by one skilled in the art that many levels of sequence identity are useful in identifying polypeptides, from other species, wherein such polypeptides have the same or similar function or activity. Useful examples of percent identities include, but are not limited to: 24%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or any integer percentage from 24% to 100% may be useful in describing the present invention, such as 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids.

The term "sequence analysis software" refers to any computer algorithm or software program that is useful for the analysis of nucleotide or amino acid sequences. "Sequence analysis software" may be commercially available or independently developed. Typical sequence analysis software will include, but is not limited to: 1.) the GCG suite of programs (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol., 215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.) Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTA program incorporating the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Within the context of this application it will be understood that where sequence analysis software is used for analysis, that the results of the analysis will be based on the "default values" of the program referenced, unless otherwise specified. As used herein "default values" will mean any set of values or parameters that originally load with the software when first initialized.

Standard recombinant DNA and molecular cloning techniques used here are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987).

High Flux of Pyruvate to 2,3-Butanediol in Lactic Acid Bacteria

The present invention discloses that a high proportion of pyruvate may be converted to 2,3-butanediol in lactic acid bacterial cells when the cells are genetically modified to be substantially free of lactate dehydrogenase activity and genetically modified to express heterologous polypeptides having butanediol dehydrogenase activity.

Lactic acid bacteria are well characterized and have been used commercially for many years for the production of a wide variety of products. A number of fermentation pathways exist in nature for the metabolism of sugars though pyruvate (see FIG. 4), however lactic acid bacteria have systems that favor the conversion of pyruvate to lactic acid via lactic acid dehydrogenase. It is an object of the present invention to maximize carbon flow from pyruvate to 2,3-butanediol for the production of 2-butanol and 2-butanone (FIGS. 4, and 1). Surprisingly, as described herein, it was found that the pathway modifications of the present invention resulted in a lactic acid host cell that, instead of producing mainly lactate with a small amount of acetoin as in cells without these genetic modifications, the modified cells produced 2,3-butanediol, ethanol, succinate, formate, lactate, and acetoin products. The amount of 2,3-butanediol produced is at least about 49 Mol % of the total of these 6 products At least about 0.4 gram of 2,3-butanediol may be produced per gram of glucose consumed.

2,3-butanediol is made from pyruvate through steps of pyruvate conversion to acetolactate, acetolactate conversion to acetoin, and acetoin conversion to 2,3-butanediol. This biosynthetic pathway is the first three steps (a, b, and i) of the pathway shown in FIG. 1, which is described further below. Activities performing the first and second conversions may be provided by endogenous host enzymes as exemplified herein, or may be provided by expression of heterologous enzymes as described further below.

Production of 2,3-butanediol may be achieved in cells that are lactic acid bacteria (LAB), due to the redirection of carbon flow from lactic acid production. LAB which may be host cells in the present disclosure include, but are not limited to, Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

In addition, it was determined that it is not necessary to provide an additional electron sink to balance redox equivalents to achieve the described flux from pyruvate to 2,3-butanediol. As lactate is the major end product for Lactobacillus plantarum, the NAD-dependent lactate dehydrogenases are major contributors to balancing redox equivalents. In the absence of the lactate dehydrogenases, it was expected that an additional electron sink would be needed to help balance redox. However, Applicants found that the co-production of ethanol and succinate by native enzymes was sufficient to balance redox equivalents to obtain the flux described herein, such that an additional electron sink was not needed.

Reduced Lactate Dehydrogenase Activity

Endogenous lactate dehydrogenase activity in lactic acid bacteria (LAB) converts pyruvate to lactate. LAB may have one or more genes, typically one, two or three genes, encoding lactate dehydrogenase. For example, Lactobacillus plantarum has three genes encoding lactate dehydrogenase which are named ldhL2 (protein SEQ ID NO:6, coding region SEQ ID NO:5), ldhD (protein SEQ ID NO:2, coding region SEQ ID NO:1), and ldhL1 (protein SEQ ID NO:4, coding region SEQ ID NO:3). Lactococcus lactis has one gene encoding lactate dehydrogenase which is named ldhL (protein SEQ ID NO:20, coding region SEQ ID NO:19), and Pediococcus pentosaceus has two genes named ldhD (protein SEQ ID NO:26, coding region SEQ ID NO:25) and ldhL (protein SEQ ID NO:28, coding region SEQ ID NO:27).

In the present LAB strains, lactate dehydrogenase activity is reduced so that the cells are substantially free of lactate dehydrogenase activity. Genetic modification is made in at least one gene encoding lactate dehydrogenase to reduce activity. When more than one lactate dehydrogenase gene is active under the growth conditions to be used, each of these active genes may be modified to reduce expression and thereby reduce or eliminate lactate dehydrogenase activity. For example, in L. plantarum ldhL1 and ldhD genes are modified. It is not necessary to modify the third gene, ldhL2, for growth in typical conditions as this gene appears to be inactive in these conditions. Typically, expression of one or more genes encoding lactate dehydrogenase is disrupted to reduce expressed enzyme activity. Examples of LAB lactate dehydrogenase genes that may be targeted for disruption are represented by the coding regions of SEQ ID NOs:1, 3, 5, 19, 21, 23, 25, 27, 29, 31, and 33 listed in Table 1. Other target genes, such as those encoding lactate dehydrogenase proteins having at least about 80-85%, 85%-90%, 90%-95%, or at least about 98% sequence identity to the lactate dehydrogenases of SEQ ID NOs:2, 4, 6, 20, 22 24, 26, 28, 30, 32, and 34 listed in Table 1 may be identified in the literature and using bioinformatics approaches, as is well known to one of ordinary skill in the art, since lactate dehydrogenases are well known. Typically BLAST (described above) searching of publicly available databases with known lactate dehydrogenase amino acid sequences, such as those provided herein, is used to identify lactate dehydrogenases, and their encoding sequences, that may be targets for disruption to reduce lactate dehydrogenase activity. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature in other LAB strains. For example each of the lactate dehydrogenase encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to: 1.) methods of nucleic acid hybridization; 2.) methods of DNA and RNA amplification, as exemplified by various uses of nucleic acid amplification technologies [e.g., polymerase chain reaction (PCR), Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or strand displacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A., 89:392 (1992)]; and 3.) methods of library construction and screening by complementation.

For example, genes encoding similar proteins or polypeptides to the lactate dehydrogenase encoding genes described herein could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired organism using methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the disclosed nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis, supra). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan (e.g., random primers DNA labeling, nick translation or end-labeling techniques), or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part of (or full-length of) the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full-length DNA fragments by hybridization under conditions of appropriate stringency.

Typically, in PCR-type amplification techniques, the primers have different sequences and are not complementary to each other. Depending on the desired test conditions, the sequences of the primers should be designed to provide for both efficient and faithful replication of the target nucleic acid. Methods of PCR primer design are common and well known in the art (Thein and Wallace, "The use of oligonucleotides as specific hybridization probes in the Diagnosis of Genetic Disorders", in Human Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp 33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in Molecular Biology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols: Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the described sequences may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the described nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3' end of the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3' or 5' end. Primers oriented in the 3' and 5' directions can be designed from the instant sequences. Using commercially available 3' RACE or 5' RACE systems (e.g., BRL, Gaithersburg, Md.), specific 3' or 5' cDNA fragments can be isolated (Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217 (1989)).

Alternatively, the described lactate dehydrogenase encoding sequences may be employed as hybridization reagents for the identification of homologs. The basic components of a nucleic acid hybridization test include a probe, a sample suspected of containing the gene or gene fragment of interest, and a specific hybridization method. Probes are typically single-stranded nucleic acid sequences that are complementary to the nucleic acid sequences to be detected. Probes are "hybridizable" to the nucleic acid sequence to be detected. The probe length can vary from 5 bases to tens of thousands of bases, and will depend upon the specific test to be done. Typically a probe length of about 15 bases to about 30 bases is suitable. Only part of the probe molecule need be complementary to the nucleic acid sequence to be detected. In addition, the complementarity between the probe and the target sequence need not be perfect. Hybridization does occur between imperfectly complementary molecules with the result that a certain fraction of the bases in the hybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and sample must be mixed under conditions that will permit nucleic acid hybridization. This involves contacting the probe and sample in the presence of an inorganic or organic salt under the proper concentration and temperature conditions. The probe and sample nucleic acids must be in contact for a long enough time that any possible hybridization between the probe and sample nucleic acid may occur. The concentration of probe or target in the mixture will determine the time necessary for hybridization to occur. The higher the probe or target concentration, the shorter the hybridization incubation time needed. Optionally, a chaotropic agent may be added. The chaotropic agent stabilizes nucleic acids by inhibiting nuclease activity. Furthermore, the chaotropic agent allows sensitive and stringent hybridization of short oligonucleotide probes at room temperature (Van Ness and Chen, Nucl. Acids Res. 19:5143-5151 (1991)). Suitable chaotropic agents include guanidinium chloride, guanidinium thiocyanate, sodium thiocyanate, lithium tetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate, potassium iodide and cesium trifluoroacetate, among others. Typically, the chaotropic agent will be present at a final concentration of about 3 M. If desired, one can add formamide to the hybridization mixture, typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, these comprise from about 20 to 60% volume, preferably 30%, of a polar organic solvent. A common hybridization solution employs about 30-50% v/v formamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 M buffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about 6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), or between 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal), polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Also included in the typical hybridization solution will be unlabeled carrier nucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g., calf thymus or salmon sperm DNA, or yeast RNA), and optionally from about 0.5 to 2% wt/vol glycine. Other additives may also be included, such as volume exclusion agents that include a variety of polar water-soluble or swellable agents (e.g., polyethylene glycol), anionic polymers (e.g., polyacrylate or polymethylacrylate) and anionic saccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats. One of the most suitable is the sandwich assay format. The sandwich assay is particularly adaptable to hybridization under non-denaturing conditions. A primary component of a sandwich-type assay is a solid support. The solid support has adsorbed to it or covalently coupled to it immobilized nucleic acid probe that is unlabeled and complementary to one portion of the sequence.

In the present LAB strains, at least one modification is engineered that results in cells substantially free of lactate dehydrogenase activity. This may be accomplished by eliminating expression of at least one endogenous gene encoding lactate dehydrogenase. Any genetic modification method known by one skilled in the art for reducing the expression of a protein may be used to alter lactate dehydrogenase expression. Methods include, but are not limited to, deletion of the entire or a portion of the lactate dehydrogenase encoding gene, inserting a DNA fragment into the lactate dehydrogenase encoding gene (in either the promoter or coding region) so that the encoded protein cannot be expressed, introducing a mutation into the lactate dehydrogenase coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the lactate dehydrogenase coding region to alter amino acids so that a non-functional protein is expressed. In addition lactate dehydrogenase expression may be blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression. All of these methods may be readily practiced by one skilled in the art making use of the known lactate dehydrogenase encoding sequences such as those of SEQ ID NOs: 1, 3, 5, 19, 21, 23, 25, 27, 29, 31, and 33.

For some methods genomic DNA sequences that surround a lactate dehydrogenase encoding sequence are useful, such as for homologous recombination-based methods. These sequences may be available from genome sequencing projects such as for Lactobacillus plantarum, which is available through the National Center for Biotechnology Information (NCBI) database, with Genbank.TM. identification gi|28376974|ref|NC.sub.--004567.1|[28376974]. Adjacent genomic DNA sequences may also be obtained by sequencing outward from a lactate dehydrogenase coding sequence using primers within the coding sequence, as well known to one skilled in the art.

A particularly suitable method for creating a genetically modified LAB strain substantially free of lactate dehydrogenase activity, as exemplified herein in Example 1, is using homologous recombination mediated by lactate dehydrogenase coding region flanking DNA sequences to delete the entire gene. The flanking sequences are cloned adjacent to each other so that a double crossover event using these flanking sequences deletes the lactate dehydrogenase coding region.

Expression of Heterologous Butanediol Dehydrogenase Activity

Lactic acid bacteria may naturally have a low amount of 2,3-butanediol synthesis, which may vary depending on the growth conditions. In the present invention, expression of heterologous butanediol dehydrogenase activity provides a pathway to 2,3-butanediol synthesis that successfully competes with other pathways that use pyruvate as an initial substrate, in the absence of lactate dehydrogenase activity. Heterologous butanediol dehydrogenase activity is expressed in a LAB cell that is substantially free of lactate dehydrogenase activity as described above.

Butanediol dehydrogenase enzymes are well-known and are described in the definitions above. The skilled person will appreciate that polypeptides having butanediol dehydrogenase activity isolated from a variety of sources will be useful in the present invention independent of sequence homology. Some examples of suitable butanediol dehydrogenase enzymes include, but are not limited to, those from Klebsiella pneumoniae (DNA: SEQ ID NO:12, protein: SEQ ID NO:13), Bacillus cereus (DNA: SEQ ID NO:63, protein: SEQ ID NO:64), and Lactococcus lactis (DNA: SEQ ID NO:65, protein: SEQ ID NO:66).

Because butanediol dehydrogenases are well known, and because of the prevalence of genomic sequencing, suitable butanediol dehydrogenases may be readily identified by one skilled in the art on the basis of sequence similarity using bioinformatics approaches. Typically BLAST (described above) searching of publicly available databases with known butanediol dehydrogenase amino acid sequences, such as those provided herein, is used to identify butanediol dehydrogenases, and their encoding sequences, that may be used in the present strains.

Examples of genes encoding butanediol dehydrogenase, which may be used to provide heterologous expression of butanediol dehydrogenase activity in the present LAB, have SEQ ID NOs: 12, 63, and 64 and are listed in Table 2. Additional butanediol dehydrogenase encoding genes that may be used for heterologous expression in LAB may be identified in the literature and in bioinformatics databases well known to the skilled person.

Encoding sequences for butanediol dehydrogenase proteins having amino acid sequence identities of at least about 70-75%, 75%-80%, 80-85%, 85%-90%, 90%-95%, or 98% sequence identity to any of the butanediol dehydrogenase proteins of SEQ ID NOs:13, 64 and 66 listed in Table 2 may be expressed in the present strains. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additionally, the sequences encoding butanediol dehydrogenases described herein or those recited in the art may be used to identify other homologs in nature. For example each of the butanediol dehydrogenase encoding nucleic acid fragments described herein may be used to isolate genes encoding homologous proteins. Isolation of homologous genes using sequence-dependent protocols is well known in the art, as described above for lactate dehydrogenase encoding nucleic acid fragments.

Expression of heterologous butanediol dehydrogenase is achieved by transforming suitable host cells with a sequence encoding a butanediol dehydrogenase protein. Typically the coding sequence is part of a chimeric gene used for transformation, which includes a promoter operably linked to the coding sequence as well as a ribosome binding site and a termination control region. A chimeric gene is heterologous even if it includes the coding sequence for a butanediol dehydrorgenase from the host cell for transformation, if the coding sequence is combined with regulatory sequences that are not native to the natural gene encoding butanediol dehydrogenase.

Codons may be optimized for expression based on codon usage in the selected host, as is known to one skilled in the art. Vectors useful for the transformation of a variety of host cells are common and described in the literature. Typically the vector contains a selectable marker and sequences allowing autonomous replication or chromosomal integration in the desired host. In addition, suitable vectors may comprise a promoter region which harbors transcriptional initiation controls and a transcriptional termination control region, between which a coding region DNA fragment may be inserted, to provide expression of the inserted coding region. Both control regions may be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions may also be derived from genes that are not native to the specific species chosen as a production host.

Initiation control regions or promoters, which are useful to drive expression of a butanediol dehydrogenase coding region in LAB are familiar to those skilled in the art. Some examples include the amy, apr, and npr promoters; nisA promoter (useful for expression Gram-positive bacteria (Eichenbaum et al. Appl. Environ. Microbiol. 64(8):2763-2769 (1998)); and the synthetic P11 promoter (useful for expression in Lactobacillus plantarum, Rud et al., Microbiology 152:1011-1019 (2006)). In addition, the ldhL1 and fabZ1 promoters of L plantarum are useful for expression of chimeric genes in LAB. The fabZ1 promoter directs transcription of an operon with the first gene, fabZ1, encoding (3R)-hydroxymyristoyl-[acyl carrier protein] dehydratase.

Termination control regions may also be derived from various genes, typically from genes native to the preferred hosts. Optionally, a termination site may be unnecessary, however, it is most preferred if included.

Vectors useful in LAB include vectors having two origins of replication and two selectable markers which allow for replication and selection in both Escherichia coli and LAB. An example is pFP996, the sequence of which is provided as SEQ ID NO:35, which is useful in L. plantarum and other LAB. Many plasmids and vectors used in the transformation of Bacillus subtilis and Streptococcus may be used generally for LAB. Non-limiting examples of suitable vectors include pAM.beta.1 and derivatives thereof (Renault et al., Gene 183:175-182 (1996); and O'Sullivan et al., Gene 137:227-231 (1993)); pMBB1 and pHW800, a derivative of pMBB1 (Wyckoff et al. Appl. Environ. Microbiol. 62:1481-1486 (1996)); pMG1, a conjugative plasmid (Tanimoto et al., J. Bacteriol. 184:5800-5804 (2002)); pNZ9520 (Kleerebezem et al., Appl. Environ. Microbiol. 63:4581-4584 (1997)); pAM401 (Fujimoto et al., Appl. Environ. Microbiol. 67:1262-1267 (2001)); and pAT392 (Arthur et al., Antimicrob. Agents Chemother. 38:1899-1903 (1994)). Several plasmids from Lactobacillus plantarum have also been reported (e.g., van Kranenburg R, Golic N, Bongers R, Leer R J, de Vos W M, Siezen R J, Kleerebezem M. Appl. Environ. Microbiol. 2005 March; 71(3): 1223-1230).

Vectors may be introduced into a host cell using methods known in the art, such as electroporation (Cruz-Rodz et al. Molecular Genetics and Genomics 224:1252-154 (1990), Bringel, et al. Appl. Microbiol. Biotechnol. 33: 664-670 (1990), Alegre et al., FEMS Microbiology letters 241:73-77 (2004)), and conjugation (Shrago et al., Appl. Environ. Microbiol. 52:574-576 (1986)). A chimeric butanediol dehydrogenase gene can also be integrated into the chromosome of LAB using integration vectors (Hols et al., Appl. Environ. Microbiol. 60:1401-1403 (1990), Jang et al., Micro. Lett. 24:191-195 (2003)).

Reducing Pyruvate Formate Lyase Activity

In addition to the modifications described above with respect to lactate dehydrogenase and butanediol dehydrogenase in the present cells, optionally these cells may further have at least one modification that reduces endogenous pyruvate formate lyase activity. Pyruvate formate lyase activity converts pyruvate to formate. Activity of pyruvate formate lyase in the cell may be reduced or eliminated. Preferably the activity is eliminated.

For expression of pyruvate formate lyase activity a gene encoding pyruvate formate lyase (pfl) and a gene encoding pyruvate formate lyase activating enzyme are required. To reduce pyruvate formate lyase activity a modification may be made in either or both of these genes. There may be one or more genes encoding each of pyruvate formate lyase and pyruvate formate lyase activating enzyme in a particular strain of LAB. For example, Lactobacillus plantarum WCFS1 contains two pfl genes (pflB1: coding region SEQ ID NO:69, protein SEQ ID NO:70; and pflB2: coding region SEQ ID NO:71, protein SEQ ID NO:72) and two pfl activating enzyme genes (pflA1: coding region SEQ ID NO:73, protein SEQ ID NO:74; and pflA2: coding region SEQ ID NO:75, protein SEQ ID NO:76), Lactobacillus plantarum PN0512 only contains one pfl gene (pflB2) and one pfl activating enzyme gene (pflA2). In one embodiment, expression is reduced for all pfl encoding genes that are active in a production host cell under the desired production conditions and/or for all pfl activating enzyme encoding genes that are active in a production host cell under the desired production conditions.

Examples of pfl genes that may be modified to reduce pyruvate formate lyase activity are represented by the coding regions of SEQ ID NOs: 39, 41, 47, and 51. Other target genes for modification include those encoding pyruvate formate lyase proteins having SEQ ID NOs:40, 42, 48, and 52 and those encoding a protein having at least about 80-85%, 85%-90%, 90%-95%, or at least about 96%, 97%, 98%, or 99% sequence identity to one of these proteins, which may be identified in the literature and using bioinformatics approaches, as is well known to the skilled person as described above for lactate dehydrogenase proteins. Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature as described above.

Examples of pfl activating enzyme genes that may be modified to reduce pyruvate formate lyase activity are represented by the coding regions of SEQ ID NOs:73, 75, 79, and 83. Other target genes for modification include those encoding pyruvate formate lyase activating enzyme proteins having SEQ ID NOs:74, 76, 80, 84 and those encoding a protein having at least about 80-85%, 85%-90%, 90%-95%, or at least about 96%, 97%, 98%, or 99% sequence identity to one of these proteins, which may be identified in the literature and using bioinformatics approaches, as is well known to the skilled person as described above for lactate dehydrogenase proteins. Additionally, the sequences described herein or those recited in the art may be used to identify other homologs in nature as described above.

Any genetic modification method known by one skilled in the art for reducing the expression of a protein may be used to alter pyruvate formate lyase activity. Methods to reduce or eliminate expression of the pyruvate formate lyase and/or pyruvate formate lyase activating enzyme genes include, but are not limited to, deletion of the entire or a portion of the gene, inserting a DNA fragment into the gene (in either the promoter or coding region) so that the encoded protein cannot be expressed or has reduced expression, introducing a mutation into the coding region which adds a stop codon or frame shift such that a functional protein is not expressed, and introducing one or more mutations into the coding region to alter amino acids so that a non-functional or reduced-functional protein is expressed. In addition expression from the target gene may be partially or substantially blocked by expression of an antisense RNA or an interfering RNA, and constructs may be introduced that result in cosuppression.

Product Biosynthesis in LAB Engineered for High Flux of Pyruvate to 2,3-butanediol

2,3-butanediol and any product that has 2,3-butanediol as a pathway intermediate may be produced with greater effectiveness (such as greater rate, titer, yield, and/or efficiency thereof) in a LAB cell disclosed herein having high flux of pyruvate to 2,3-butanediol. Such products include, but are not limited to, 2,3-butanediol, 2-butanone, and 2-butanol.

A biosynthetic pathway for synthesis of 2,3-butanediol, 2-butanone and 2-butanol is disclosed in US Patent Pub No. US20070292927A1, which is herein incorporated by reference. A diagram of the disclosed 2,3-butanediol, 2-butanone and 2-butanol biosynthetic pathway is provided in FIG. 1 therein. 2,3-butanediol is the product of the first three steps, which are listed below. 2-Butanone is the product made when the last depicted step of converting 2-butanone to 2-butanol is omitted. Production of 2-butanone or 2-butanol in a strain disclosed herein benefits from increased production of 2,3-butanediol. As described in US Patent Pub No. US20070292927A1, steps in the biosynthetic pathway include conversion of: pyruvate to acetolactate (see FIG. 1, step a therein) as catalyzed for example by acetolactate synthase (ALS) known by the EC number 2.2.1.69; acetolactate to acetoin (see FIG. 1, step b therein) as catalyzed for example by acetolactate decarboxylase; acetoin to 2,3-butanediol (see FIG. 2, step i therein) as catalyzed for example by butanediol dehydrogenase; 2,3-butanediol to 2-butanone (see FIG. 2, step j therein) as catalyzed for example by diol dehydratase or glycerol dehydratase; and 2-butanone to 2-butanol (see FIG. 2, step f therein) as catalyzed for example by butanol dehydrogenase.

Genes that may be used to engineer expression of these enzymes are described in US Patent Pub No. 20070292927A1. Alternatively endogenous enzymes in LAB may perform some pathway steps, such as acetolactate synthase and acetolactate decarboxylase. The use in this pathway of the butanediol dehydratase from Roseburia inulinivorans, RdhtA, (protein SEQ ID NO:16, coding region SEQ ID NO:15) is disclosed in US Patent Pub No. US 20090155870A1. This enzyme is used in conjunction with the butanediol dehydratase reactivase from Roseburia inulinivorans, RdhtB, (protein SEQ ID NO:18, coding region SEQ ID NO:17). This butanediol dehydratase is desired in many hosts because it does not require coenzyme B.sub.12.

Some representative ALS enzymes that may be used include those encoded by alsS of Bacillus and budB of Klebsiella (Gollop et al., J. Bacteriol. 172(6):3444-3449 (1990); Holtzclaw et al., J. Bacteriol. 121(3):917-922 (1975)). ALS from Bacillus subtilis (DNA: SEQ ID NO:85; protein: SEQ ID NO:86), from Klebsiella pneumoniae (DNA: SEQ ID NO:88; protein: SEQ ID NO:89), and from Lactococcus lactis (DNA: SEQ ID NO:90; protein: SEQ ID NO:91) are provided herein. Additional Als coding regions and encoded proteins that may be used include those from Staphylococcus aureus (DNA: SEQ ID NO:92; protein: SEQ ID NO:93), Listeria monocytogenes (DNA: SEQ ID NO:94; protein: SEQ ID NO:95), Streptococcus mutans (DNA: SEQ ID NO:96; protein: SEQ ID NO:97), Streptococcus thermophilus (DNA: SEQ ID NO:98; protein: SEQ ID NO:99), Vibrio angustum (DNA: SEQ ID NO:100; protein: SEQ ID NO:101), and Bacillus cereus (DNA: SEQ ID NO:102; protein: SEQ ID NO:103). Any Als gene that encodes an acetolactate synthase having at least about 80-85%, 85%-90%, 90%-95%, or at least about 96%, 97%, or 98% sequence identity to any one of those with SEQ ID NOs:86, 89, 91, 93, 95, 97, 99, 101, or 103 that converts pyruvate to acetolactate may be used. Identities are based on the Clustal W method of alignment using the default parameters of GAP PENALTY=10, GAP LENGTH PENALTY=0.1, and Gonnet 250 series of protein weight matrix.

Additionally, U.S. patent application Ser. No. 12/477,942 provides a phylogenetic tree depicting acetolactate synthases that are the 100 closest neighbors of the B. subtilis AlsS sequence, any of which may be used. Additional Als sequences that may be used in the present strains may be identified in the literature and in bioinformatics databases as is well known to the skilled person. Identification of coding and/or protein sequences using bioinformatics is typically through BLAST (described above) searching of publicly available databases with known Als encoding sequences or encoded amino acid sequences, such as those provided herein. Identities are based on the Clustal W method of alignment as specified above. Additionally, the sequences listed herein or those recited in the art may be used to identify other homologs in nature as described above.

Useful for the last step of converting 2-butanone to 2-butanol is a new butanol dehydrogenase isolated from an environmental isolate of a bacterium identified as Achromobacter xylosoxidans that is disclosed in U.S. patent application Ser. No. 12/430,356 (DNA: SEQ ID NO:9, protein SEQ ID NO:10).

Chimeric genes that include the coding regions for enzymes of the pathway, or desired portion of the pathway, may be constructed and used in vectors as described above for butanediol dehydrogenase, and as disclosed in US 20070292927A1, to engineer 2,3-butanediol, 2-butanone or 2-butanol producing cells.

Growth for Production

Recombinant LAB cells disclosed herein may be used for fermentation production of 2,3-butanediol, 2-butanol or 2-butanone. The recombinant cells are grown in fermentation media which contains suitable carbon substrates. Suitable substrates may include but are not limited to monosaccharides such as glucose and fructose, oligosaccharides such as lactose or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt.

Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of monosaccharides including C5 sugars such as xylose and arabinose. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassaya, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Patent Pub No. 2007/0031918A1, which is herein incorporated by reference. Biomass refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.

In addition to an appropriate carbon source, fermentation media must contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathway necessary for 2,3-butanediol, 2-butanol or 2-butanone production. Typically cells are grown at a temperature in the range of about 25.degree. C. to about 40.degree. C. in an appropriate medium. Suitable growth media are common commercially prepared media such as Bacto Lactobacilli MRS broth or Agar (Difco), Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth. Other defined or synthetic growth media may also be used, and the appropriate medium for growth of the particular bacterial strain will be known by one skilled in the art of microbiology or fermentation science. The use of agents known to modulate catabolite repression directly or indirectly, e.g., cyclic adenosine 2':3'-monophosphate, may also be incorporated into the fermentation medium.

Suitable pH ranges for the fermentation are between pH 5.0 to pH 9.0, where pH 6.0 to pH 8.0 is preferred as the initial condition.

Fermentations may be performed under aerobic or anaerobic conditions, where anaerobic or microaerobic conditions are preferred.

2,3-butanediol, 2-butanol or 2-butanone may be produced using a batch method of fermentation. A classical batch fermentation is a closed system where the composition of the medium is set at the beginning of the fermentation and not subject to artificial alterations during the fermentation. A variation on the standard batch system is the fed-batch system. Fed-batch fermentation processes are also suitable in the present invention and comprise a typical batch system with the exception that the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is apt to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the media. Batch and fed-batch fermentations are common and well known in the art and examples may be found in Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992), herein incorporated by reference.

2,3-butanediol, 2-butanol or 2-butanone may also be produced using continuous fermentation methods. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned media is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation allows for the modulation of one factor or any number of factors that affect cell growth or end product concentration. Methods of modulating nutrients and growth factors for continuous fermentation processes as well as techniques for maximizing the rate of product formation are well known in the art of industrial microbiology and a variety of methods are detailed by Brock, supra.

It is contemplated that the production of 2,3-butanediol, 2-butanol or 2-butanone may be practiced using either batch, fed-batch or continuous processes and that any known mode of fermentation would be suitable. Additionally, it is contemplated that cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for 2,3-butanediol, 2-butanol or 2-butanone production.

Methods for 2,3-butanediol, 2-butanol or 2-butanone Isolation from the Fermentation Medium

Bioproduced 2,3-butanediol, 2-butanol or 2-butanone may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see for example, Durre, Appl. Microbiol. Biotechnol. 49:639-648 (1998), Groot et al., Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol 2,3-butanediol, 2-butanol or 2-butanone may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

EXAMPLES

The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

The meaning of abbreviations used is as follows: "min" means minute(s), "h" means hour(s), "sec` means second(s), ".mu.l" means microliter(s), "ml" means milliliter(s), "L" means liter(s), "nm" means nanometer(s), "mm" means millimeter(s), "cm" means centimeter(s), ".mu.m" means micrometer(s), "mM" means millimolar, "M" means molar, "mmol" means millimole(s), ".mu.mole" means micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "mg" means milligram(s), "rpm" means revolutions per minute, "w/v" means weight/volume, "OD" means optical density, and "OD600" means optical density measured at a wavelength of 600 nm.

General Methods:

Standard recombinant DNA and molecular cloning techniques used in the Examples are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, by T. J. Silhavy, M. L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1984, and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, N.Y., 1987. Additional methods used in the Examples are described in manuals including Advanced Bacterial Genetics (Davis, Roth and Botstein, Cold Spring Harbor Laboratory, 1980), Experiments with Gene Fusions (Silhavy, Berman and Enquist, Cold Spring Harbor Laboratory, 1984), Experiments in Molecular Genetics (Miller, Cold Spring Harbor Laboratory, 1972) Experimental Techniques in Bacterial Genetics (Maloy, in Jones and Bartlett, 1990), and A Short Course in Bacterial Genetics (Miller, Cold Spring Harbor Laboratory 1992).

Example 1

Construction of the Lactobacillus plantarum PN0512 .DELTA.ldhD.DELTA.ldhL1 Strain PNP0001

The purpose of this example is to describe the construction of a Lactobacillus plantarum PN0512 strain that is deleted for the two genes that encode the major lactate dehydrogenases. The major end product of fermentation in Lactobacillus plantarum is lactic acid. Pyruvate is converted to lactate by the action of two lactate dehydrogenases encoded by the ldhD and ldhL1 genes. A double deletion of ldhD and ldhL1 was made in Lactobacillus plantarum PN0512 (ATCC strain #PTA-7727).

Gene knockouts were constructed using a process based on a two-step homologous recombination procedure to yield unmarked gene deletions (Ferain et al., 1994, J. Bact. 176:596). The procedure utilized a shuttle vector, pFP996 (SEQ ID NO:35). pFP996 is a shuttle vector for gram-positive bacteria. It can replicate in both E. coli and gram-positive bacteria. It contains the origins of replication from pBR322 (nucleotides #2628 to 5323) and pE194 (nucleotides #43 to 2627). pE194 is a small plasmid isolated originally from a gram positive bacterium, Staphylococcus aureus (Horinouchi and Weisblum J. Bacteriol. (1982) 150(2):804-814). In pFP996, the multiple cloning sites (nucleotides #1 to 50) contain restriction sites for EcoRI, BglII, XhoI, SmaI, ClaI, KpnI, and HindIII. There are two antibiotic resistance markers; one is for resistance to ampicillin and the other for resistance to erythromycin. For selection purposes, ampicillin was used for transformation in E. coli and erythromycin was used for selection in L. plantarum.

Two segments of DNA, each containing 900 to 1200 by of sequence either upstream or downstream of the intended deletion, were cloned into the plasmid to provide the regions of homology for the two genetic cross-overs. Cells were grown for an extended number of generations (30-50) to allow for the cross-over events to occur. The initial cross-over (single cross-over) integrated the plasmid into the chromosome by homologous recombination through one of the two homology regions on the plasmid. The second cross-over (double cross-over) event yielded either the wild-type sequence or the intended gene deletion. A cross-over between the sequences that led to the initial integration event would yield the wild-type sequence, while a cross-over between the other regions of homology would yield the desired deletion. The second cross-over event was screened for by antibiotic sensitivity. Single and double cross-over events were analyzed by PCR and DNA sequencing.

All restriction enzymes, DNA modifying enzymes and Phusion High-Fidelity PCR Master Mix were purchased from NEB Inc. (Ipswich, Ma). PCR SuperMix and Platinum PCR SuperMix High Fidelity were purchased from Invitrogen Corp (Carlsbad, Calif.). DNA fragments were gel purified using Zymoclean.TM. Gel DNA Recovery Kit (Zymo Research Corp, Orange, Calif.) or Qiaquick PCR Purification Kit (Qiagen Inc., Valencia, Calif.). Plasmid DNA was prepared with QIAprep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.). Oligoucleotides were synthesized by Sigma-Genosys (Woodlands, Tex.) or Invitrogen Corp (Carlsbad, Calif.). L. plantarum PN0512 genomic DNA was prepared with MasterPure DNA Purification Kit (Epicentre, Madison, Wis.).

Lactobacillus plantarum PN0512 was transformed by the following procedure: 5 ml of Lactobacilli MRS medium (Accumedia, Neogen Corporation, Lansing, Mich.) containing 1% glycine (Sigma-Aldrich, St. Louis, Mo.) was inoculated with PN0512 cells and grown overnight at 30.degree. C. 100 ml MRS medium with 1% glycine was inoculated with overnight culture to an OD600 of 0.1 and grown to an OD600 of 0.7 at 30.degree. C. Cells were harvested at 3700.times.g for 8 min at 4.degree. C., washed with 100 ml cold 1 mM MgCl.sub.2 (Sigma-Aldrich, St. Louis, Mo.), centrifuged at 3700.times.g for 8 min at 4.degree. C., washed with 100 ml cold 30% PEG-1000 (Sigma-Aldrich, St. Louis, Mo.), recentrifuged at 3700.times.g for 20 min at 4.degree. C., then resuspended in 1 ml cold 30% PEG-1000. 60 .mu.l cells were mixed with .about.100 ng plasmid DNA in a cold 1 mm gap electroporation cuvette and electroporated in a BioRad Gene Pulser (Hercules, Calif.) at 1.7 kV, 25 .mu.F, and 400.OMEGA.. Cells were resuspended in 1 ml MRS medium containing 500 mM sucrose (Sigma-Aldrich, St. Louis, Mo.) and 100 mM MgCl.sub.2, incubated at 30.degree. C. for 2 hrs, plated on MRS medium plates containing 1 or 2 .mu.g/ml of erythromycin (Sigma-Aldrich, St. Louis, Mo.), then placed in an anaerobic box containing a Pack-Anaero sachet (Mitsubishi Gas Chemical Co., Tokyo, Japan) and incubated at 30.degree. C.

.DELTA.ldhD

The knockout cassette to delete the ldhD gene was created by amplifying from PN0512 genomic DNA an upstream flanking region with primers Top D F1 (SEQ ID NO:39) containing an EcoRI site and Top D R1 (SEQ ID NO:40). The downstream homology region including part of the coding sequence of ldhD was amplified with primers Bot D F2 (SEQ ID NO:41) and Bot D R2 (SEQ ID NO:42) containing an XhoI site. The two homology regions were joined by PCR SOE as follows. The 0.9 kbp upstream and downstream PCR products were gel-purified. The PCR products were mixed in equal amounts in a PCR reaction and re-amplified with primers Top D F1 and Bot D R2. The final 1.8 kbp PCR product was gel-purified and TOPO cloned into pCR4BluntII-TOPO (Invitrogen) to create vector pCRBluntII::ldhD. To create the integration vector carrying the internal deletion of the ldhD gene, pFP996 was digested with EcoRI and XhoI and the 5311-bp fragment gel-purified. Vector pCRBluntII::ldhD was digested with EcoRI and XhoI and the 1.8 kbp fragment gel-purified. The ldhD knockout cassette and vector were ligated using T4 DNA ligase, resulting in vector pFP996::ldhD ko.

Electrocompetent Lactobacillus plantarum PN0512 cells were prepared, transformed with pFP996::ldhD ko, and plated on MRS containing 1 .mu.g/ml of erythromycin. To obtain the single-crossover event (sco), transformants were passaged for approximately 50 generations in MRS medium at 37.degree. C. After growth, aliquots were plated for single colonies on MRS containing 1 .mu.g/ml of erythromycin. The erythromycin-resistant colonies were screened by PCR amplification with primers ldhD Seq F1 (SEQ ID NO:43) and D check R (SEQ ID NO:44) to distinguish between wild-type and clones carrying the sco event. To obtain clones with a double crossover, the sco strains were passaged for approximately 30 generations in MRS medium with 20 mM D, L-lactate (Sigma, St. Louis, Mo.) at 37.degree. C. and then plated for single colonies on MRS with lactate. Colonies were picked and patched onto MRS with lactate and MRS with lactate containing 1 .mu.g/ml of erythromycin to find colonies sensitive to erythromycin. Sensitive colonies were screened by PCR amplification using primer D check R (SEQ ID NO:44) and D check F3 (SEQ ID NO:45). Wild-type colonies gave a 3.2 kbp product and deletion clones, called PN0512.DELTA.ldhD, gave a 2.3 kbp PCR product.

.DELTA.ldhD.DELTA.ldhL1

A deletion of the ldhL1 gene was made in the PN0512.DELTA.ldhD strain background in order to make a double .DELTA.ldhL1.DELTA.ldhD deletion strain. The knockout cassette to delete the ldhL1 gene was amplified from PN0512 genomic DNA. The ldhL1 left homologous arm was amplified using primers oBP31 (SEQ ID NO:46) containing a BglII restriction site and oBP32 (SEQ ID NO:47) containing an XhoI restriction site. The ldhL1 right homologous arm was amplified using primers oBP33 (SEQ ID NO:48) containing an XhoI restriction site and oBP34 (SEQ ID NO:49) containing an XmaI restriction site. The ldhL1 left homologous arm was cloned into the BglII/XhoI sites and the ldhL1 right homologous arm was cloned into the XhoI/XmaI sites of pFP996pyrF.DELTA.erm, a derivative of pFP996. pFP996pyrF.DELTA.erm contains the pyrF sequence (SEQ ID NO:7) encoding orotidine-5'-phosphate decarboxylase from Lactobacillus plantarum PN0512 in place of the erythromycin coding region in pFP996. The plasmid-borne pyrF gene, in conjunction with the chemical 5-fluoroorotic acid in a .DELTA.pyrF strain, can be used as an effective counter-selection method in order to isolate the second homologous crossover. The XmaI fragment containing the ldhL1 homologous arms was isolated following XmaI digestion and cloned into the XmaI restriction site of pFP996, yielding a 900 by left homologous region and a 1200 by right homologous region resulting in vector pFP996-ldhL1-arms.

PN0512.DELTA.ldhD was transformed with pFP996-ldhL1-arms and grown at 30.degree. C. in Lactobacilli MRS medium with lactate (20 mM) and erythromycin (1 .mu.g/ml) for approximately 10 generations. Transformants were then grown under non-selective conditions at 37.degree. C. for about 50 generations by serial inoculations in MRS+ lactate before cultures were plated on MRS containing lactate and erythromycin (1 .mu.g/ml). Isolates were screened by colony PCR for a single crossover using chromosomal specific primer oBP49 (SEQ ID NO:53) and plasmid specific primer oBP42 (SEQ ID NO:54). Single crossover integrants were grown at 37.degree. C. for approximately 40 generations by serial inoculations under non-selective conditions in MRS with lactate before cultures were plated on MRS medium with lactate. Isolates were patched to MRS with lactate plates, grown at 37.degree. C., and then patched onto MRS plates with lactate and erythromycin (1 .mu.g/ml). Erythromycin sensitive isolates were screened by colony PCR for the presence of a wild-type or deletion second crossover using chromosomal specific primers oBP49 (SEQ ID NO:53) and oBP56 (SEQ ID NO:55). A wild-type sequence yielded a 3505 by product and a deletion sequence yielded a 2545 by product. The deletions were confirmed by sequencing the PCR product and absence of plasmid was tested by colony PCR with primers oBP42 (SEQ ID NO:54) and oBP57 (SEQ ID NO:58).

The Lactobacillus plantarum PN0512 double ldhDldhL1 deletion strain was designated PNP0001. The .DELTA.ldhD deletion included 83 by upstream of where the ldhD start codon was through amino acid 279 of 332. The .DELTA.ldhL1 deletion included the fMet through the final amino acid.

Example 2

Product Analysis of a Lactobacillus plantarum Strain Deleted for the Two Lactate Dehydrogenases, LdhD and LdhL1

The purpose of this example is to demonstrate the products produced by the Lactobacillus plantarum PN0512 double ldhDldhL1 deletion strain compared to the wild-type strain.

Strains PN0512 (wild-type) and PNP0001 (.DELTA.ldhD.DELTA.ldhL1) were grown in rich medium, Lactobacilli MRS medium (Accumedia, Neogen Corporation, Lansing, Mich.), at 30.degree. C. without shaking under anaerobic conditions in an anaerobic chamber (Coy Laboratories Inc., Grass Lake, Mich.). Both cultures were grown to a similar OD600 about 8.5. PNP0001 grew at a rate that was approximately 2.5 times slower than the wild-type PN0512. In order to reach a similar OD600, strain PN0512 was grown for 16 hours and strain PNP0001 was grown for 41 hours. Cultures were centrifuged at 3700.times.g for 10 minutes at 4.degree. C. and culture supernatants were filtered through a 0.2 .mu.m filter (Pall Life Sciences, Ann Arbor, Mich.). The filtered supernatants were analyzed by HPLC with column Shodex SUGAR SH1011 (Showa Denko K.K., Kawasaki, Japan) and refractive index detection for levels of glucose, citrate, acetate, lactate, acetoin, ethanol, succinate, and formate.

Results in FIG. 2 show the consumption of the medium constituents and the products that were formed. 71% of the 114 mM glucose was consumed in the PN0512 culture and 158 mM lactic acid was produced. Significant amounts of other products were not detected. 99% of the glucose, as well as 100% of the 12 mM citrate and 76% of the 70 mM acetate was consumed in the PNP0001 culture. PNP0001 produced only 1 mM lactate. Instead, the main products were acetoin (102 mM) and ethanol (93 mM), along with succinate (28 mM) and formate (31 mM). These data demonstrated that the .DELTA.ldhD and .DELTA.ldhL1 deletions effectively eliminated major production of lactic acid and led to a mixed fermentation product profile.

Example 3

Construction of Plasmids for the Production of meso-2,3-butanediol

The purpose of this example is to describe the construction of a plasmid for expression of a heterologous butanediol dehydrogenase. The ldhL1 promoter region (SEQ ID NO:8) from L. plantarum PN0512 was amplified with primers AA135 (SEQ ID NO:61), containing EcoRI, SpeI, and AflII sites, and AA136 (SEQ ID NO:62), containing an XhoI site, from PN0512 genomic DNA using Phusion High-Fidelity PCR Master Mix. The resulting PCR fragment and pFP996 were ligated after digestion with EcoRI and XhoI to create vector pFP996PldhL1 (SEQ ID NO:36).

A secondary alcohol dehydrogenase encoded by the Achromobacter xylosoxidans sadB gene (coding region SEQ ID NO:9 and protein SEQ ID NO:10) was disclosed in U.S. patent application Ser. No. 12/430,356. The sadB coding region was amplified with primers oBP112 (SEQ ID NO:50), containing XhoI, NheI, and EcoRV sites along with a ribosome binding site (SEQ ID NO:51), and oBP113 (SEQ ID NO:52), containing a KpnI site, from vector pRS426::FBA-budC+GPM-sadB using Phusion High-Fidelity PCR Master Mix. pRS426 is a yeast shuttle vector (American Type Culture Collection, Rockville, Md.), which contains an E. coli replication origin (e.g., pMB1), a yeast 2.mu. origin of replication, and Ura3 marker for nutritional selection. pRS426::FBA-budC+GPM-sadB contains the FBA promoter (SEQ ID NO:11) from the S. cerevisiae fructose 1,6-bisphosphate aldolase gene operably linked to the budC coding region for butanediol dehydrogenase from Klebsiella pneumonia (coding region SEQ ID NO:12). In addition it has the yeast GPM1 promoter (SEQ ID NO:14) operably linked to the Achromobacter xylosoxidans sadB coding region (SEQ ID NO:9). The construction of pRS426::FBA-budC+GPM-sadB is described in Example 3 of U.S. patent application Ser. No. 12/477,942, which is herein incorporated by reference.

The sadB coding region PCR fragment and pFP996PldhL1 were ligated after digestion with XhoI and KpnI to create vector pFP996PldhL1-sadB. The Klebsiella pneumoniae budC coding region for butanediol dehydrogenase (SEQ ID NO:12) was amplified with primers oBP114 (SEQ ID NO:56), containing a NheI site and a ribosome binding site, and oBP115 (SEQ ID NO:57), containing an EcoRV site, from vector pRS426::FBA-budC+GPM-sadB using Phusion High-Fidelity PCR Master Mix. The resulting PCR fragment and pFP996PldhL1-sadB were ligated after digestion with NheI and EcoRV to create vector pFP996PldhL1-budC-sadB (SEQ ID NO:37). The sadB gene in vector pFP996PldhL1-budC-sadB was deleted to create vector pFP996PldhL1-budC (SEQ ID NO:38). Vector pFP996PldhL1-budC-sadB was digested with EcoRV and HindIII, the HindIII site was filled in with T4 DNAP, and then the plasmid was re-ligated. Candidates were screened by colony PCR with primers oBP42 (SEQ ID NO:54) and oBP57 (SEQ ID NO:58) for plasmids that did not contain the sadB gene and then sequenced.

Example 4

Production of meso-2,3-butanediol Using a Recombinant Lactobacillus plantarum Strain Grown in Rich Medium

The purpose of this example is to demonstrate the production of meso-2,3-butanediol using a recombinant Lactobacillus plantarum strain containing an engineered pathway in rich medium. Specifically, a Lactobacillus plantarum strain deleted for the two endogenous lactate dehydrogenases, LdhD and LdhL1, and containing a plasmid, pFP996PldhL1-budC-sadB, expressing the Klebsiella pneumoniae budC coding region for butanediol dehydrogenase was grown in MRS medium. The first two enzymes for the butanediol pathway, acetolactate synthase and acetolactate decarboxylase, were provided by native expression from the chromosome. sadB encodes a butanol dehydrogenase that in the presence of 2-butanone would provide an electorn sink that could be required to balance redox equivalents for 2,3-butanediol production.

Wild-type Lactobacillus plantarum strain PN0512 and strain PNP0001 were transformed with plasmid pFP996PldhL1-budC-sadB. Strains were transformed as in Example 1, except glycine was omitted from the medium for strain PNP0001. The resulting PNP0001/pFP996PldhL1-budC-sadB strain was designated PNP0002 and the PN0512/pFP996-budC-sadB strain designated BP134. Strains were grown in MRS medium with 0.5% 2-butanone. Strains containing plasmids were grown in medium also containing 2 .mu.g/ml of erythromycin.

145 ml of medium was inoculated with strains PNP0001, PNP0001/pFP996PldhL1-budC-sadB (PNP0002), or PN0512/pFP996PldhL1-budC-sadB (BP134) from overnight cultures at a dilution of 1:145 in 175 ml sealed serum bottles. Cultures were grown at 30.degree. C. for 24 hours without shaking. Strain BP134 reached an OD600 6.5, strain PNP0001 an OD600 8.1, and strain PNP0002 an OD600 6.2. The cultures were started at a higher inoculum so there was a shorter lag and fewer doublings to get to saturation, to reduce the difference in growth that was observed in Example 2. Samples of the cultures were centrifuged at 3700.times.g for 10 minutes at 4.degree. C. and the supernatants filtered through a 0.2 .mu.m filter (Pall Life Sciences, Ann Arbor, Mich.). The filtered supernatants were analyzed by HPLC with column Shodex SUGAR SH1011 (Showa Denko K.K., Kawasaki, Japan) and refractive index detection for levels of glucose, citrate, acetate, lactate, acetoin, meso-2,3-butanediol, ethanol, succinate, and formate.

Results in FIG. 3 show the consumption of the medium constituents and the products that were formed. Strain BP134 consumed 84% of the glucose, 64% of the citrate, and no acetate. This strain produced, similar to the wild-type strain without the plasmid, almost entirely lactic acid, 172 mM. Strain PNP0001 consumed 91% of the glucose, 100% of the citrate, and 82% of the acetate. As in example 2, the main products of strain PNP0001 were acetoin (86 mM) and ethanol (73 mM), along with succinate (21 mM) and formate (8 mM). Strain PNP0002 consumed 92% of the glucose, 100% of the citrate, and 53% of the acetate. In contrast to strain PNP0001, no acetoin was detected for strain PNP0002. Instead, the main product was meso-2,3-butanediol (78 mM), along with ethanol (54 mM), succinate (19 mM), and formate (7 mM). Meso-2,3-butanediol accounted for 49 Mol % of the measured products. These data showed that with the presence of the heterologous budC expressing plasmid in the double ldh deletion strain, acetoin was converted to meso-2,3-butanediol when cells were grown in rich medium. The titer of meso-2,3-butanediol was 7.0 g/L with a yield of 0.41 g/g of glucose consumed.

Example 5

Production of meso-2,3-butanediol Using a Recombinant Lactobacillus plantarum Strain Containing Vector pFP996PldhL1-budC-sadB Grown in Synthetic Medium with Glucose or Sucrose

The purpose of this example is to demonstrate the production of meso-2,3-butanediol using a recombinant Lactobacillus plantarum strain containing an engineered pathway in synthetic medium. Specifically, a Lactobacillus plantarum strain deleted for the two endogenous lactate dehydrogenases, LdhD and LdhL1, and containing a plasmid, pFP996PldhL1-budC-sadB, expressing the Klebsiella pneumoniae budC coding region for butanediol dehydrogenase was grown in synthetic medium with glucose or sucrose. The first two enzymes for the butanediol pathway, acetolactate synthase and acetolactate decarboxylase, were provided by native expression from the chromosome.

Strain PNP0001/pFP996PldhL1-budC-sadB (PNP0002) was grown in a synthetic medium with 20 mM glucose or sucrose and 2 .mu.g/ml erythromycin. The synthetic medium consisted of: 10 mM ammonium sulfate, 100 mM MES pH6, 5 mM potassium phosphate pH 6, 1% S10 metal mix, 20 mM glucose or sucrose, 0.5% yeast extract, 0.01% casamino acids, and 10 mM ammonium citrate. 100% S10 metal mix consists of 200 mM MgCl.sub.2, 70 mM CaCl.sub.2, 5 mM MnCl.sub.2, 100 .mu.M FeCl.sub.3, 100 .mu.M ZnCl.sub.2, 172 .mu.M CuSO.sub.4, 253 .mu.M CoCl.sub.2, 242 .mu.M NaMoO.sub.4, and 200 .mu.M thiamine hydrochloride. All medium constituents were purchased from Sigma-Aldrich (St. Louis, Mo.). 25 ml of medium was inoculated with PNP0002 and grown at 30.degree. C. overnight without shaking in an anaerobic box containing a Pack-Anaero sachet (Mitsubishi Gas Chemical Co., Tokyo, Japan) to an OD600 0.72 (glucose) or 0.88 (sucrose). Overnight cultures were centrifuged for 5 minutes at 5 k RPM and then resuspended in fresh medium at a final dilution of 1:10. 25 ml of culture was grown in an anaerobic box with a Pack-Anaero sachet at 30.degree. C. without shaking for 28 hours to an OD600 3.18 (glucose) or 4.52 (sucrose). Samples were centrifuged and supernatants filtered through a 0.2 .mu.m filter (Pall Life Sciences, Ann Arbor, Mich.). The filtered supernatants were analyzed by GC with column HP-Innowax Polyethylene Glycol (19091N-113, Agilent Technologies, Santa Clara, Calif.) and flame ionization detection for levels of meso-2,3-butanediol, acetoin, and ethanol. The results in Table 5 show that meso-2,3-butanediol accounted for greater than 50% of the two main products, meso-2,3-butanediol and ethanol, similar to results obtained with rich medium.

TABLE-US-00005 TABLE 5 Production of meso-2,3-butanediol, acetoin, and ethanol by PNP0001/pFP996PldhL1-budC-sadB grown in synthetic medium with glucose or sucrose. Concentration (mM) Culture meso-2,3-butanediol Acetoin ethanol Glucose 12.9 2.4 10.9 Sucrose 25.5 3.1 10.0

These data demonstrated that a recombinant Lactobacillus plantarum strain deleted for the ldhD and ldhL1 genes and containing a plasmid expressing the heterologous gene budC produced meso-2,3-butanediol when cells were grown in synthetic medium with either glucose or sucrose as the fermentable sugar.

Production of 2,3-butanediol without 2-butanone in the medium indicated that the additional electron sink was not needed to provide redox balance for the flux described.

Example 6

Production of meso-2,3-butanediol Using a Recombinant Lactobacillus plantarum Strain Containing Vector pFP996PldhL1-budC Grown in Synthetic Medium with Sucrose

The purpose of this example is to demonstrate the production of meso-2,3-butanediol using a recombinant Lactobacillus plantarum strain containing an engineered pathway in synthetic medium. Specifically, a Lactobacillus plantarum strain deleted for the two endogenous lactate dehydrogenases, LdhD and LdhL1, and containing a plasmid, pFP996PldhL1-budC, expressing the Klebsiella pneumoniae budC coding region for butanediol dehydrogenase was grown in synthetic medium with sucrose. The first two enzymes for the butanediol pathway, acetolactate synthase and acetolactate decarboxylase, were provided by native expression from the chromosome. Since Example 5 showed that no additional redox balancing electron sink was needed, sadB expression was not included.

Strain PNP0001 was transformed, as in Example 1 except glycine was omitted, with plasmids pFP996PldhL1 and pFP996PldhL1-budC. Strains PNP0001/pFP996PldhL1 and PNP0001/pFP996PldhL1-budC were grown overnight in Lactobacilli MRS medium with 2 .mu.g/ml erythromycin at 30.degree. C. in an anaerobic chamber (Coy Laboratories Inc., Grass Lake, Mich.). Vials containing synthetic medium, which had been deoxygenated overnight in an anaerobic chamber, were inoculated with overnight culture to an OD600 of about 0.02 and sealed in the anaerobic chamber. The synthetic medium consisted of: 10 mM ammonium sulfate, 100 mM MES pH6, 5 mM potassium phosphate pH6, 1% S10 metal mix, 20 mM sucrose, 0.5% yeast extract, 0.01% casamino acids, 10 mM ammonium citrate, and 2 .mu.g/ml erythromycin. Cultures were grown at 30.degree. C. without shaking for 48 hours to an OD600 about 2.3. Samples of the cultures were centrifuged at 3700.times.g for 10 minutes at 4.degree. C. and the supernatants filtered through a 0.2 .mu.m filter (Pall Life Sciences, Ann Arbor, Mich.). The filtered supernatants were analyzed by GC with column HP-Innowax Polyethylene Glycol (19091N-113, Agilent Technologies, Santa Clara, Calif.) and flame ionization detection for levels of meso-2,3-butanediol, acetoin, and ethanol.

Results in Table 5 show the production of meso-2,3-butanediol, acetoin, and ethanol for strain PNP0001/pFP996PldhL1-budC grown in synthetic medium with sucrose. The amount of meso-2,3-butanediol produced by this strain is comparable to PNP0001 with vector pFP996PldhL1-budC-sadB (Example 5).

TABLE-US-00006 TABLE 5 Production of meso-2,3-butanediol, acetoin, and ethanol by PNP0001/pFP996PldhL1 and PNP0001/pFP996PldhL1-budC grown in synthetic medium with sucrose. Concentration (mM) Strain meso-2,3-butanediol acetoin ethanol PNP0001/pFP996PldhL1 0.5 26.2 24.9 PNP0001/pFP996PldhL1-budC 33.3 2.7 18.1

Example 7

Prophetic

Production of 2-butanol by a Recombinant L. plantraum Strain Expressing B.sub.12-Independent Diol Dehydratase

A vector expressing butanediol dehydrogenase encoded by the Klebsiella pneumoniae budC gene, secondary alcohol dehydrogenase encoded by the Achromobacter xylosoxidans sadB gene, and coenzyme B.sub.12-independent (S-adenosylmethionine-dependent) butanediol dehydratase and its associated reactivase encoded by the Roseburia inulinivorans rdhtA (DNA SEQ ID NO: 15; protein SEQ ID NO:16) and rdhtB (DNA SEQ ID NO: 17; protein SEQ ID NO:18) genes respectively, is constructed. The Roseburia inulinivorans coenzyme B.sub.12-independent propanediol dehydratase and reactivase are disclosed in US Patent Pub No. US20090155870A1. Therein the sequences encoding rdhtA and rdhtB were synthesized as one DNA fragment (SEQ ID NO:67) by standard methods and cloned into an E. coli vector (by DNA2.0, Inc., Menlo Park, Calif.) resulting in pJ206::rdhtAB.

The Roseburia inulinivorans rdhtA and rdhtB coding regions are amplified with primers rdhtAB-up (SEQ ID NO:59) and rdhtAB-down (SEQ ID NO:60), each containing a BsrGI restriction site, from vector pJ206::rdhtAB. The resulting PCR fragment and pFP996PldhL1-budC-sadB are ligated after digestion with BsrGI and used to transform E. coli TOP10 cells. Plasmids that have the rdhtAB coding regions in the same orientation as budC and sadB are identified by PCR with primers rhdtAB-up (SEQ ID NO:59) and oBP42 (SEQ ID NO:54) and the resulting, correctly oriented clone is named pFP996PldhL1-budC-sadB-rdhtAB.

Strain PNP0001 is transformed with vector pFP996PldhL1-budC-sadB-rdhtAB as described in Example 1, except glycine is omitted from the medium. MRS medium containing 2 .mu.g/ml erythromycin is inoculated with strain PNP0001/pFP996PldhL1-budC-sadB-rdhtAB and grown overnight at 30.degree. C. in an anaerobic chamber. Vials containing MRS medium with 2 .mu.g/ml erythromycin, which is deoxygenated overnight in an anaerobic chamber, are inoculated with overnight culture at a 1:100 dilution and sealed in the anaerobic chamber. Cultures are grown at 30.degree. C. without shaking for 48 hours. The culture supernatant is tested and 2-butanol is detected by HPLC or GC.

Example 8

Construction of the Lactobacillus plantarum PN0512 .DELTA.ldhD.DELTA.ldhL1.DELTA.pflB2A 2::alsS(o) Strain

The purpose of this example is to describe the construction of a Lactobacillus plantarum strain in the PN0512.DELTA.ldhD.DELTA.ldhL1 strain background that is deleted for the genes pflB2, encoding formate C-acetyltransferase (pyruvate formate lyase), and pflA2, encoding the formate C-acetyltransferase activating enzyme, and thus does not contain formate C-acetyltransferase activity. Whereas Lactobacillus plantarum WCFS1 contains two genes encoding formate C-acetyltransferase and two genes encoding formate C-acetyltransferase activating enzyme, Lactobacillus plantarum PN0512 only contains one gene encoding formate C-acetyltransferase and one gene encoding formate C-acetyltransferase activating enzyme. A gene (alsS), codon optimized for expression in Lactobacillus plantarum, encoding the Bacillus subtilis acetolactate synthase enzyme was integrated in place of the deleted pflB2A2 genes.

The pflB2A2 gene knockout and alsS gene integration were constructed using the two-step homologous recombination procedure described above. The knockout deleted the C-terminal 351 amino acids (nucleotides 1204 through 2256 of the coding sequence) of PflB2 and the entire coding sequence of pflA2. The deleted sequence was replaced with a stop codon, in frame with the truncated pflB2, followed by a ribosome binding sequence and Bacillus subtilis alsS gene codon optimized for expression in Lactobacillus plantarum.

The knockout/integration vector was constructed in plasmid pFP996 as follows. The homologous arms to delete the pflB2A2 genes were amplified from PN0512 genomic DNA. The pflB2A2 left homologous arm was amplified using primers oBP309 (SEQ ID NO:104) containing an XhoI restriction site and oBP310 (SEQ ID NO:105) containing a stop codon (complement of TAA) and XmaI restriction site. The pflB2A2 right homologous arm was amplified using primers oBP271 (SEQ ID NO:106) containing a KpnI restriction site and oBP272 (SEQ ID NO:107) containing a BsrGI restriction site. The pflB2A2 left homologous arm was cloned into the XhoI/XmaI sites and the pflB2A2 right homologous arm was cloned into the KpnI/BsrGI sites of pFP996 to create pFP996-pflB2A2arms. The Bacillus subtilis alsS gene codon optimized for expression in Lactobacillus plantarum (SEQ ID NO:87; synthesized by Genscript Corp, Piscataway, N.J.) was amplified using primers oBP282 (SEQ ID NO:108) containing an XmaI restriction site and oBP283 (SEQ ID NO:109) containing a KpnI restriction site. The codon optimized alsS gene was cloned into the XmaI/KpnI sites of pFP996-pflB2A2arms to create pFP996-pflB2A2arms-als(o).

PN0512 .DELTA.ldhD.DELTA.ldhL1 was transformed with pFP996-pflB2A2arms-als(o) as above, except competent cells were prepared in the absence of glycine, and transformants were selected on MRS plates containing 1 .mu.g/ml erythromycin. A transformant was grown at 30.degree. C. for about 35 generations by serial inoculations in MRS before cultures were plated on MRS containing erythromycin (1 .mu.g/ml). Isolates were screened by colony PCR for a single crossover using chromosomal specific primer oAA227 (SEQ ID NO:110) and plasmid specific primer oBP42 (SEQ ID NO:54). A single crossover integrant was grown at 37.degree. C. for approximately 35 generations by serial inoculations in MRS before cultures were plated on MRS medium. Erythromycin sensitive isolates were screened by colony PCR for the presence of a wild-type or deletion/integration second crossover using als(o) specific primer oAA228 (SEQ ID NO:111) and chromosomal specific primer oBP280 (SEQ ID NO:112). The deletion/integration strain PN0512 .DELTA.ldhD.DELTA.ldhL1.DELTA.pflB2A2::als(o).sup.+, named BP556, was confirmed by sequencing the PCR product amplified with chromosomal specific primers oBP278 (SEQ ID NO:113) and oBP280 (SEQ ID NO:112).

Example 9

Production of meso-2,3-butanediol Using a Recombinant Lactobacillus plantarum Strain Lacking Both Lactate Dehydrogenase Activity and Formate C-Acetyltransferase Activity Grown in Rich Medium

The purpose of this example is to demonstrate the production of meso-2,3-butanediol using a recombinant Lactobacillus plantarum strain containing an engineered pathway in rich medium. Specifically, a Lactobacillus plantarum strain deleted for the two endogenous lactate dehydrogenases, LdhD and LdhL1, deleted for the formate C-acetyltransferase, PflB2, and containing a plasmid, pFP996PldhL1-budC, expressing the Klebsiella pneumoniae budC coding region for butanediol dehydrogenase was grown in MRS medium. The second enzyme for the butanediol pathway, acetolactate decarboxylase, was provided by native expression from the chromosome. The first enzyme for the butanediol pathway, acetolactate synthase, was provided by native expression from the chromosome and the heterologous Bacillus subtilis alsS gene integrated into the pflB2A2 locus.

Strain BP556 was transformed as in Example 1, except glycine was omitted, with plasmid pFP996PldhL1-budC. Strains PNP0001/pFP996PldhL1-budC and BP556/pFP996PldhL1-budC were grown overnight in Lactobacilli MRS medium with 2 .mu.g/ml erythromycin at 30.degree. C. Overnight cultures were used to inoculate 5 ml MRS medium with 2 .mu.g/ml erythromycin in 15 ml screw cap tubes. Cultures were grown at 30.degree. C. without shaking in an anaerobic box containing a Pack-Anaero sachet (Mitsubishi Gas Chemical Co., Tokyo, Japan) for 24 hours to an OD600 about 6.5. Samples of the cultures were centrifuged at 3700.times.g for 10 minutes at 4.degree. C. and the supernatants filtered through a 0.2 .mu.m filter (Pall Life Sciences, Ann Arbor, Mich.). The filtered supernatants were analyzed by HPLC with column Shodex SUGAR SH1011 (Showa Denko K.K., Kawasaki, Japan) and refractive index detection. Greater than 99% of the glucose was consumed in both cultures. The pflB2A2 deletion led to no detectable levels of formate for strain BP556/pFP996PldhL1-budC, whereas strain PNP0001/pFP996PldhL1-budC produced 20 mM formate. Production of meso-2,3-butanediol increased 12% for BP556/pFP996PldhL1-budC (92 mM) compared to PNP0001/pFP996PldhL1-budC (82 mM).

SEQUENCE LISTINGS

1

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 113 <210> SEQ ID NO 1 <211> LENGTH: 999 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 1 atgaaaatta ttgcatatgc tgtacgtgat gacgaacgtc cattcttcga tacttggatg 60 aaagaaaacc cagatgttga agttaaatta gttccagaat tacttactga agacaacgtt 120 gacttagcta aaggcttcga cggtgccgat gtataccaac aaaaggacta tactgctgaa 180 gtattgaaca agttagccga cgaaggggtt aagaacatct ctcttcgtaa cgttggtgtt 240 gataacttgg acgttcctac tgttaaagca cgtggcttaa acatttctaa cgtacctgca 300 tactcaccaa atgcgattgc tgaattatca gtaacgcaat tgatgcaatt attacgtcaa 360 accccattgt tcaataagaa gttagctaag caagacttcc gttgggcacc agatattgcc 420 aaggaattaa acaccatgac tgttggtgtt atcggtactg gtcggattgg ccgtgctgcc 480 atcgatattt tcaaaggctt cggcgctaag gttatcggtt acgatgttta ccggaatgct 540 gaacttgaaa aggaaggcat gtacgttgac accttggacg aattatacgc ccaagctgat 600 gttatcacgt tacacgttcc tgcattgaag gataactacc acatgttgaa tgcggatgcc 660 ttcagcaaga tgaaagatgg cgcctacatc ttgaactttg ctcgtgggac actcatcgat 720 tcagaagact tgatcaaagc cttagacagt ggcaaagttg ccggtgccgc tcttgatacg 780 tatgaatacg aaactaagat cttcaacaaa gaccttgaag gtcaaacgat tgatgacaag 840 gtcttcatga acttgttcaa ccgcgacaat gttttgatta caccacatac ggctttctac 900 actgaaactg ccgttcacaa catggtgcac gtttcaatga acagtaacaa acaattcatc 960 gaaactggta aagctgatac gcaagttaag tttgactaa 999 <210> SEQ ID NO 2 <211> LENGTH: 332 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 2 Met Lys Ile Ile Ala Tyr Ala Val Arg Asp Asp Glu Arg Pro Phe Phe 1 5 10 15 Asp Thr Trp Met Lys Glu Asn Pro Asp Val Glu Val Lys Leu Val Pro 20 25 30 Glu Leu Leu Thr Glu Asp Asn Val Asp Leu Ala Lys Gly Phe Asp Gly 35 40 45 Ala Asp Val Tyr Gln Gln Lys Asp Tyr Thr Ala Glu Val Leu Asn Lys 50 55 60 Leu Ala Asp Glu Gly Val Lys Asn Ile Ser Leu Arg Asn Val Gly Val 65 70 75 80 Asp Asn Leu Asp Val Pro Thr Val Lys Ala Arg Gly Leu Asn Ile Ser 85 90 95 Asn Val Pro Ala Tyr Ser Pro Asn Ala Ile Ala Glu Leu Ser Val Thr 100 105 110 Gln Leu Met Gln Leu Leu Arg Gln Thr Pro Leu Phe Asn Lys Lys Leu 115 120 125 Ala Lys Gln Asp Phe Arg Trp Ala Pro Asp Ile Ala Lys Glu Leu Asn 130 135 140 Thr Met Thr Val Gly Val Ile Gly Thr Gly Arg Ile Gly Arg Ala Ala 145 150 155 160 Ile Asp Ile Phe Lys Gly Phe Gly Ala Lys Val Ile Gly Tyr Asp Val 165 170 175 Tyr Arg Asn Ala Glu Leu Glu Lys Glu Gly Met Tyr Val Asp Thr Leu 180 185 190 Asp Glu Leu Tyr Ala Gln Ala Asp Val Ile Thr Leu His Val Pro Ala 195 200 205 Leu Lys Asp Asn Tyr His Met Leu Asn Ala Asp Ala Phe Ser Lys Met 210 215 220 Lys Asp Gly Ala Tyr Ile Leu Asn Phe Ala Arg Gly Thr Leu Ile Asp 225 230 235 240 Ser Glu Asp Leu Ile Lys Ala Leu Asp Ser Gly Lys Val Ala Gly Ala 245 250 255 Ala Leu Asp Thr Tyr Glu Tyr Glu Thr Lys Ile Phe Asn Lys Asp Leu 260 265 270 Glu Gly Gln Thr Ile Asp Asp Lys Val Phe Met Asn Leu Phe Asn Arg 275 280 285 Asp Asn Val Leu Ile Thr Pro His Thr Ala Phe Tyr Thr Glu Thr Ala 290 295 300 Val His Asn Met Val His Val Ser Met Asn Ser Asn Lys Gln Phe Ile 305 310 315 320 Glu Thr Gly Lys Ala Asp Thr Gln Val Lys Phe Asp 325 330 <210> SEQ ID NO 3 <211> LENGTH: 963 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 3 ttgtcaagca tgccaaatca tcaaaaagtt gtgttagtcg gcgacggcgc tgttggttct 60 agttacgctt ttgccatggc acaacaagga attgctgaag aatttgtaat tgtcgatgtt 120 gttaaagatc ggacaaaggg tgacgccctt gatcttgaag acgcccaagc attcaccgct 180 cccaagaaga tttactcagg cgaatattca gattgtaagg acgctgactt agttgttatt 240 acagccggtg cgcctcaaaa gcctggtgaa tcacgtttag acttagttaa caagaattta 300 aatatcctat catccattgt caaaccagtt gttgactccg gctttgacgg catcttctta 360 gttgctgcta accctgttga catcttaact tacgctactt ggaaattctc aggtttccca 420 aaggatcgtg tcattggttc agggacttcc ttagactctt cacgtttacg cgttgcgtta 480 ggcaaacaat tcaatgttga tcctcgttcc gttgatgctt acatcatggg tgaacacggt 540 gattctgaat ttgctgctta ctcaactgca accatcggga cacgtccagt tcgcgatgtc 600 gctaaggaac aaggcgtttc tgacgaagat ttagccaagt tagaagacgg tgttcgtaac 660 aaagcttacg acatcatcaa cttgaagggt gccacgttct acggtatcgg gactgcttta 720 atgcggattt ccaaagccat tttacgtgat gaaaatgccg ttttaccagt aggtgcctac 780 atggacggcc aatacggctt aaacgacatt tatatcggga ctccggctgt gattggtgga 840 actggtttga aacaaatcat cgaatcacca ctttcagctg acgaactcaa gaagatgcaa 900 gattccgccg caactttgaa aaaagtgctt aacgacggtt tagctgaatt agaaaataaa 960 taa 963 <210> SEQ ID NO 4 <211> LENGTH: 320 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 4 Met Ser Ser Met Pro Asn His Gln Lys Val Val Leu Val Gly Asp Gly 1 5 10 15 Ala Val Gly Ser Ser Tyr Ala Phe Ala Met Ala Gln Gln Gly Ile Ala 20 25 30 Glu Glu Phe Val Ile Val Asp Val Val Lys Asp Arg Thr Lys Gly Asp 35 40 45 Ala Leu Asp Leu Glu Asp Ala Gln Ala Phe Thr Ala Pro Lys Lys Ile 50 55 60 Tyr Ser Gly Glu Tyr Ser Asp Cys Lys Asp Ala Asp Leu Val Val Ile 65 70 75 80 Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Ser Arg Leu Asp Leu Val 85 90 95 Asn Lys Asn Leu Asn Ile Leu Ser Ser Ile Val Lys Pro Val Val Asp 100 105 110 Ser Gly Phe Asp Gly Ile Phe Leu Val Ala Ala Asn Pro Val Asp Ile 115 120 125 Leu Thr Tyr Ala Thr Trp Lys Phe Ser Gly Phe Pro Lys Asp Arg Val 130 135 140 Ile Gly Ser Gly Thr Ser Leu Asp Ser Ser Arg Leu Arg Val Ala Leu 145 150 155 160 Gly Lys Gln Phe Asn Val Asp Pro Arg Ser Val Asp Ala Tyr Ile Met 165 170 175 Gly Glu His Gly Asp Ser Glu Phe Ala Ala Tyr Ser Thr Ala Thr Ile 180 185 190 Gly Thr Arg Pro Val Arg Asp Val Ala Lys Glu Gln Gly Val Ser Asp 195 200 205 Glu Asp Leu Ala Lys Leu Glu Asp Gly Val Arg Asn Lys Ala Tyr Asp 210 215 220 Ile Ile Asn Leu Lys Gly Ala Thr Phe Tyr Gly Ile Gly Thr Ala Leu 225 230 235 240 Met Arg Ile Ser Lys Ala Ile Leu Arg Asp Glu Asn Ala Val Leu Pro 245 250 255 Val Gly Ala Tyr Met Asp Gly Gln Tyr Gly Leu Asn Asp Ile Tyr Ile 260 265 270 Gly Thr Pro Ala Val Ile Gly Gly Thr Gly Leu Lys Gln Ile Ile Glu 275 280 285 Ser Pro Leu Ser Ala Asp Glu Leu Lys Lys Met Gln Asp Ser Ala Ala 290 295 300 Thr Leu Lys Lys Val Leu Asn Asp Gly Leu Ala Glu Leu Glu Asn Lys 305 310 315 320 <210> SEQ ID NO 5 <211> LENGTH: 930 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 5 atggataaga agcaacgcaa agtcgtaatt gttggtgatg gctcggtggg ttcatcattt 60 gccttttcat tggtccaaaa ttgcgcccta gatgaactcg ttatcgttga cttggttaaa 120 acgcacgcag agggggacgt taaggatttg gaagatgttg ccgcctttac gaatgcgacc 180 aacattcata ccggtgaata tgcggatgcg cgtgatgctg acatcgttgt cattacggct 240 ggtgtgcctc gtaagcctgg tgagagtcgt ttagatttga ttaaccgcaa tacgaagatt 300 ctggaatcca tcgtcaaacc agtggttgcg agtggtttta atggttgctt cgttatctca 360 agtaatcccg tcgatatttt gacttcgatg acgcaacgtt tatccggttt tccacggcat 420

cgggtcattg gtaccgggac ttccttggat acggcgcggt tacgggtcgc cttggctcag 480 aagttgaatg ttgccaccac tgcagttgat gctgcggtac ttggagaaca tggtgatagt 540 tccatcgtta attttgatga aattatgatc aatgctcagc ccttaaagac ggtcacaacg 600 gtcgatgatc agttcaaagc tgaaatcgag caagctgttc gtggtaaagg tggtcaaatc 660 attagtcaga agggggccac gttctatggg gtcgccgtta gtttgatgca aatctgccga 720 gcaattttga acgatgaaaa tgctgagttg attgtctccg ccgctttgtc tggtcaatat 780 ggcattaacg atttgtactt ggggtcaccc gccattatta accgcaacgg gctccaaaaa 840 gtgatcgaag ctgagctatc agatgatgag cgtgcccgga tgcaacattt cgcagccaag 900 atgctgacca tgatgaatgt ggcatcataa 930 <210> SEQ ID NO 6 <211> LENGTH: 309 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 6 Met Asp Lys Lys Gln Arg Lys Val Val Ile Val Gly Asp Gly Ser Val 1 5 10 15 Gly Ser Ser Phe Ala Phe Ser Leu Val Gln Asn Cys Ala Leu Asp Glu 20 25 30 Leu Val Ile Val Asp Leu Val Lys Thr His Ala Glu Gly Asp Val Lys 35 40 45 Asp Leu Glu Asp Val Ala Ala Phe Thr Asn Ala Thr Asn Ile His Thr 50 55 60 Gly Glu Tyr Ala Asp Ala Arg Asp Ala Asp Ile Val Val Ile Thr Ala 65 70 75 80 Gly Val Pro Arg Lys Pro Gly Glu Ser Arg Leu Asp Leu Ile Asn Arg 85 90 95 Asn Thr Lys Ile Leu Glu Ser Ile Val Lys Pro Val Val Ala Ser Gly 100 105 110 Phe Asn Gly Cys Phe Val Ile Ser Ser Asn Pro Val Asp Ile Leu Thr 115 120 125 Ser Met Thr Gln Arg Leu Ser Gly Phe Pro Arg His Arg Val Ile Gly 130 135 140 Thr Gly Thr Ser Leu Asp Thr Ala Arg Leu Arg Val Ala Leu Ala Gln 145 150 155 160 Lys Leu Asn Val Ala Thr Thr Ala Val Asp Ala Ala Val Leu Gly Glu 165 170 175 His Gly Asp Ser Ser Ile Val Asn Phe Asp Glu Ile Met Ile Asn Ala 180 185 190 Gln Pro Leu Lys Thr Val Thr Thr Val Asp Asp Gln Phe Lys Ala Glu 195 200 205 Ile Glu Gln Ala Val Arg Gly Lys Gly Gly Gln Ile Ile Ser Gln Lys 210 215 220 Gly Ala Thr Phe Tyr Gly Val Ala Val Ser Leu Met Gln Ile Cys Arg 225 230 235 240 Ala Ile Leu Asn Asp Glu Asn Ala Glu Leu Ile Val Ser Ala Ala Leu 245 250 255 Ser Gly Gln Tyr Gly Ile Asn Asp Leu Tyr Leu Gly Ser Pro Ala Ile 260 265 270 Ile Asn Arg Asn Gly Leu Gln Lys Val Ile Glu Ala Glu Leu Ser Asp 275 280 285 Asp Glu Arg Ala Arg Met Gln His Phe Ala Ala Lys Met Leu Thr Met 290 295 300 Met Asn Val Ala Ser 305 <210> SEQ ID NO 7 <211> LENGTH: 710 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 7 atgaagcgac caattatcat tgcgttagat tttcccaccg ccgaacgggc cttagctttt 60 ttagaccaat ttccggctga tttacatgtc actgtcaaaa tcggcatgga gttattttat 120 gcagcgggac cgagtattgt gacggacgtg caagctcgcg gccatgcggt tttcttagat 180 ttgaaactac atgatattcc caataccgtc gaatccgcaa tgcgggtgat cgggcggtta 240 ggggtaacct atacgacggt tcatgctgcg ggtgggcacg tgatgctttc agccgccaaa 300 cgaggattgg tcgcgggtgc aatggccgct ggagtcactg cccccaagtt attagcgatt 360 acgcagttaa cttcgactaa tcaagctatt ttgaatcagg accagcaaat catgggaacg 420 gttcgggcga gtgtcgtgca ttatgccaaa ctagcacggg cgagtgactg tgatggcgtc 480 atttgttccg cccaagaagt tcaggcgatt catacggccg tcggtgctga ttttctcgga 540 attacgccgg gaattcggcc agcgtcggcg cagtcagatg accagcaacg ggtgatgaca 600 ccggctgccg ctgctaaggc tgggagcaac ggtctcgtca tcgggcggcc aattacgcag 660 gctgcagaac cagttcaagc ttaccgagat attatgacag aatggagtaa 710 <210> SEQ ID NO 8 <211> LENGTH: 252 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 8 taagtcgtat tggcaccact actcacaccg tgaccgacgc gcccgccagt caagtgttca 60 aaagttagcg tttattaagt gcgataagta taccacaaag ggcttattga cgcccgccaa 120 agggttttgc ggacattgtt aataattgta ttaaaagcat gctcaatcta acacttattt 180 tgcacaaaca tggtatactt taaccgtaaa aactaaattt tcactacgag aggatgactt 240 attttgtcaa gc 252 <210> SEQ ID NO 9 <211> LENGTH: 1047 <212> TYPE: DNA <213> ORGANISM: Achromobacter xylosoxidans <400> SEQUENCE: 9 atgaaagctc tggtttatca cggtgaccac aagatctcgc ttgaagacaa gcccaagccc 60 acccttcaaa agcccacgga tgtagtagta cgggttttga agaccacgat ctgcggcacg 120 gatctcggca tctacaaagg caagaatcca gaggtcgccg acgggcgcat cctgggccat 180 gaaggggtag gcgtcatcga ggaagtgggc gagagtgtca cgcagttcaa gaaaggcgac 240 aaggtcctga tttcctgcgt cacttcttgc ggctcgtgcg actactgcaa gaagcagctt 300 tactcccatt gccgcgacgg cgggtggatc ctgggttaca tgatcgatgg cgtgcaggcc 360 gaatacgtcc gcatcccgca tgccgacaac agcctctaca agatccccca gacaattgac 420 gacgaaatcg ccgtcctgct gagcgacatc ctgcccaccg gccacgaaat cggcgtccag 480 tatgggaatg tccagccggg cgatgcggtg gctattgtcg gcgcgggccc cgtcggcatg 540 tccgtactgt tgaccgccca gttctactcc ccctcgacca tcatcgtgat cgacatggac 600 gagaatcgcc tccagctcgc caaggagctc ggggcaacgc acaccatcaa ctccggcacg 660 gagaacgttg tcgaagccgt gcataggatt gcggcagagg gagtcgatgt tgcgatcgag 720 gcggtgggca taccggcgac ttgggacatc tgccaggaga tcgtcaagcc cggcgcgcac 780 atcgccaacg tcggcgtgca tggcgtcaag gttgacttcg agattcagaa gctctggatc 840 aagaacctga cgatcaccac gggactggtg aacacgaaca cgacgcccat gctgatgaag 900 gtcgcctcga ccgacaagct tccgttgaag aagatgatta cccatcgctt cgagctggcc 960 gagatcgagc acgcctatca ggtattcctc aatggcgcca aggagaaggc gatgaagatc 1020 atcctctcga acgcaggcgc tgcctga 1047 <210> SEQ ID NO 10 <211> LENGTH: 348 <212> TYPE: PRT <213> ORGANISM: Achromobacter xylosoxidans <400> SEQUENCE: 10 Met Lys Ala Leu Val Tyr His Gly Asp His Lys Ile Ser Leu Glu Asp 1 5 10 15 Lys Pro Lys Pro Thr Leu Gln Lys Pro Thr Asp Val Val Val Arg Val 20 25 30 Leu Lys Thr Thr Ile Cys Gly Thr Asp Leu Gly Ile Tyr Lys Gly Lys 35 40 45 Asn Pro Glu Val Ala Asp Gly Arg Ile Leu Gly His Glu Gly Val Gly 50 55 60 Val Ile Glu Glu Val Gly Glu Ser Val Thr Gln Phe Lys Lys Gly Asp 65 70 75 80 Lys Val Leu Ile Ser Cys Val Thr Ser Cys Gly Ser Cys Asp Tyr Cys 85 90 95 Lys Lys Gln Leu Tyr Ser His Cys Arg Asp Gly Gly Trp Ile Leu Gly 100 105 110 Tyr Met Ile Asp Gly Val Gln Ala Glu Tyr Val Arg Ile Pro His Ala 115 120 125 Asp Asn Ser Leu Tyr Lys Ile Pro Gln Thr Ile Asp Asp Glu Ile Ala 130 135 140 Val Leu Leu Ser Asp Ile Leu Pro Thr Gly His Glu Ile Gly Val Gln 145 150 155 160 Tyr Gly Asn Val Gln Pro Gly Asp Ala Val Ala Ile Val Gly Ala Gly 165 170 175 Pro Val Gly Met Ser Val Leu Leu Thr Ala Gln Phe Tyr Ser Pro Ser 180 185 190 Thr Ile Ile Val Ile Asp Met Asp Glu Asn Arg Leu Gln Leu Ala Lys 195 200 205 Glu Leu Gly Ala Thr His Thr Ile Asn Ser Gly Thr Glu Asn Val Val 210 215 220 Glu Ala Val His Arg Ile Ala Ala Glu Gly Val Asp Val Ala Ile Glu 225 230 235 240 Ala Val Gly Ile Pro Ala Thr Trp Asp Ile Cys Gln Glu Ile Val Lys 245 250 255 Pro Gly Ala His Ile Ala Asn Val Gly Val His Gly Val Lys Val Asp 260 265 270 Phe Glu Ile Gln Lys Leu Trp Ile Lys Asn Leu Thr Ile Thr Thr Gly 275 280 285 Leu Val Asn Thr Asn Thr Thr Pro Met Leu Met Lys Val Ala Ser Thr 290 295 300 Asp Lys Leu Pro Leu Lys Lys Met Ile Thr His Arg Phe Glu Leu Ala 305 310 315 320 Glu Ile Glu His Ala Tyr Gln Val Phe Leu Asn Gly Ala Lys Glu Lys 325 330 335

Ala Met Lys Ile Ile Leu Ser Asn Ala Gly Ala Ala 340 345 <210> SEQ ID NO 11 <211> LENGTH: 643 <212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 11 gaaatgaata acaatactga cagtactaaa taattgccta cttggcttca catacgttgc 60 atacgtcgat atagataata atgataatga cagcaggatt atcgtaatac gtaatagttg 120 aaaatctcaa aaatgtgtgg gtcattacgt aaataatgat aggaatggga ttcttctatt 180 tttccttttt ccattctagc agccgtcggg aaaacgtggc atcctctctt tcgggctcaa 240 ttggagtcac gctgccgtga gcatcctctc tttccatatc taacaactga gcacgtaacc 300 aatggaaaag catgagctta gcgttgctcc aaaaaagtat tggatggtta ataccatttg 360 tctgttctct tctgactttg actcctcaaa aaaaaaaaat ctacaatcaa cagatcgctt 420 caattacgcc ctcacaaaaa cttttttcct tcttcttcgc ccacgttaaa ttttatccct 480 catgttgtct aacggatttc tgcacttgat ttattataaa aagacaaaga cataatactt 540 ctctatcaat ttcagttatt gttcttcctt gcgttattct tctgttcttc tttttctttt 600 gtcatatata accataacca agtaatacat attcaaatct aga 643 <210> SEQ ID NO 12 <211> LENGTH: 771 <212> TYPE: DNA <213> ORGANISM: Klebsiella pneumoniae <400> SEQUENCE: 12 atgaaaaaag tcgcacttgt taccggcgcc ggccagggga ttggtaaagc tatcgccctt 60 cgtctggtga aggatggatt tgccgtggcc attgccgatt ataacgacgc caccgccaaa 120 gcggtcgcct cggaaatcaa ccaggccggc ggacacgccg tggcggtgaa agtggatgtc 180 tccgaccgcg atcaggtatt tgccgccgtt gaacaggcgc gcaaaacgct gggcggcttc 240 gacgtcatcg tcaataacgc cggtgtggca ccgtctacgc cgatcgagtc cattaccccg 300 gagattgtcg acaaagtcta caacatcaac gtcaaagggg tgatctgggg tattcaggcg 360 gcggtcgagg cctttaagaa agaggggcac ggcgggaaaa tcatcaacgc ctgttcccag 420 gccggccacg tcggcaaccc ggagctggcg gtgtatagct ccagtaaatt cgcggtacgc 480 ggcttaaccc agaccgccgc tcgcgacctc gcgccgctgg gcatcacggt caacggctac 540 tgcccgggga ttgtcaaaac gccaatgtgg gccgaaattg accgccaggt gtccgaagcc 600 gccggtaaac cgctgggcta cggtaccgcc gagttcgcca aacgcatcac tctcggtcgt 660 ctgtccgagc cggaagatgt cgccgcctgc gtctcctatc ttgccagccc ggattctgat 720 tacatgaccg gtcagtcgtt gctgatcgac ggcgggatgg tatttaacta a 771 <210> SEQ ID NO 13 <211> LENGTH: 256 <212> TYPE: PRT <213> ORGANISM: Klebsiella pneumoniae <400> SEQUENCE: 13 Met Lys Lys Val Ala Leu Val Thr Gly Ala Gly Gln Gly Ile Gly Lys 1 5 10 15 Ala Ile Ala Leu Arg Leu Val Lys Asp Gly Phe Ala Val Ala Ile Ala 20 25 30 Asp Tyr Asn Asp Ala Thr Ala Lys Ala Val Ala Ser Glu Ile Asn Gln 35 40 45 Ala Gly Gly His Ala Val Ala Val Lys Val Asp Val Ser Asp Arg Asp 50 55 60 Gln Val Phe Ala Ala Val Glu Gln Ala Arg Lys Thr Leu Gly Gly Phe 65 70 75 80 Asp Val Ile Val Asn Asn Ala Gly Val Ala Pro Ser Thr Pro Ile Glu 85 90 95 Ser Ile Thr Pro Glu Ile Val Asp Lys Val Tyr Asn Ile Asn Val Lys 100 105 110 Gly Val Ile Trp Gly Ile Gln Ala Ala Val Glu Ala Phe Lys Lys Glu 115 120 125 Gly His Gly Gly Lys Ile Ile Asn Ala Cys Ser Gln Ala Gly His Val 130 135 140 Gly Asn Pro Glu Leu Ala Val Tyr Ser Ser Ser Lys Phe Ala Val Arg 145 150 155 160 Gly Leu Thr Gln Thr Ala Ala Arg Asp Leu Ala Pro Leu Gly Ile Thr 165 170 175 Val Asn Gly Tyr Cys Pro Gly Ile Val Lys Thr Pro Met Trp Ala Glu 180 185 190 Ile Asp Arg Gln Val Ser Glu Ala Ala Gly Lys Pro Leu Gly Tyr Gly 195 200 205 Thr Ala Glu Phe Ala Lys Arg Ile Thr Leu Gly Arg Leu Ser Glu Pro 210 215 220 Glu Asp Val Ala Ala Cys Val Ser Tyr Leu Ala Ser Pro Asp Ser Asp 225 230 235 240 Tyr Met Thr Gly Gln Ser Leu Leu Ile Asp Gly Gly Met Val Phe Asn 245 250 255 <210> SEQ ID NO 14 <211> LENGTH: 753 <212> TYPE: DNA <213> ORGANISM: Saccharomyces cerevisiae <400> SEQUENCE: 14 gcatgcttgc atttagtcgt gcaatgtatg actttaagat ttgtgagcag gaagaaaagg 60 gagaatcttc taacgataaa cccttgaaaa actgggtaga ctacgctatg ttgagttgct 120 acgcaggctg cacaattaca cgagaatgct cccgcctagg atttaaggct aagggacgtg 180 caatgcagac gacagatcta aatgaccgtg tcggtgaagt gttcgccaaa cttttcggtt 240 aacacatgca gtgatgcacg cgcgatggtg ctaagttaca tatatatata tatagccata 300 gtgatgtcta agtaaccttt atggtatatt tcttaatgtg gaaagatact agcgcgcgca 360 cccacacaca agcttcgtct tttcttgaag aaaagaggaa gctcgctaaa tgggattcca 420 ctttccgttc cctgccagct gatggaaaaa ggttagtgga acgatgaaga ataaaaagag 480 agatccactg aggtgaaatt tcagctgaca gcgagtttca tgatcgtgat gaacaatggt 540 aacgagttgt ggctgttgcc agggagggtg gttctcaact tttaatgtat ggccaaatcg 600 ctacttgggt ttgttatata acaaagaaga aataatgaac tgattctctt cctccttctt 660 gtcctttctt aattctgttg taattacctt cctttgtaat tttttttgta attattcttc 720 ttaataatcc aaacaaacac acatattaca ata 753 <210> SEQ ID NO 15 <211> LENGTH: 2532 <212> TYPE: DNA <213> ORGANISM: Roseburia inulinivorans <400> SEQUENCE: 15 atgggcaatt acgattcaac accgatagct aaaagtgata ggattaaaag attggttgat 60 catttgtatg ctaaaatgcc tgaaattgag gccgctagag cagagctaat tactgaatcc 120 tttaaggcca ccgaaggtca acctgttgtt atgagaaagg ctagagcttt tgaacatata 180 ctaaagaatt tgccaattat cataagacca gaagaactga ttgttggctc aactacaatt 240 gcccctagag gttgccaaac gtatccagaa ttctcatacg agtggttaga ggctgaattt 300 gaaactgtcg aaacgcgttc agctgaccca ttttatattt cagaagaaac gaagaaacgt 360 ttgctggctg ccgatgctta ttggaaaggt aaaacaacct cagagttggc aacttcatat 420 atggccccag aaactctaag agccatgaag cataacttct tcacccctgg aaactacttc 480 tacaatggtg tcggtcatgt cacagttcaa tatgaaacag tattagcaat cggcttgaat 540 ggagtaaaag agaaggttag gaaagagatg gagaattgtc attttggtga tgccgattat 600 agtacaaaga tgtgtttctt ggagagcatt ttaatatcgt gtgatgccgt aatcacttat 660 gctaatagat atgccaagat ggccgaggaa atggctgaaa aagaaacaga tgctgcaagg 720 aggcaagaac tattaacaat cgccagggtt tgcaaaaacg ttcctgaatt cccagccgaa 780 agcttccagg aggcctgcca atccttttgg ttcatacaac aagtgcttca aattgaatcc 840 agtggtcatt caatttcccc aggtagattt gatcaatata tgtatcctta ttacgaaaag 900 gatttaaagg aaggtagctt aactagggaa tatgctcagg aactgatcga ttgtatctgg 960 gttaagttaa atgatctgaa taagtgcagg gatgctgcct ctgctgaggg ctttgcagga 1020 tattccttat ttcaaaactt aatcgttggg ggccaaacgg ttcaaggaag ggacgccacc 1080 aatgatttga gttttatgtg tatcacggca tctgaacacg tctttttacc gatgccgtcg 1140 ttgtctataa gagtttggca tggtagttcc aaagcactgc ttatgagagc agctgaattg 1200 actagaaccg gtataggctt acctgcttat tacaatgatg aagtcatcat accagctttg 1260 gtgcataggg gtgctactat ggatgaagca agaaattaca acataatagg atgtgtcgaa 1320 ccgcaggttc ctggtaaaac tgatggctgg cacgatgcag cattctttaa catgtgcaga 1380 cctttggaaa tggtgtttag taatggttat gataacggtg aaattgcatc tatacaaact 1440 ggtaacgtag aatcttttca gagttttgat gagtttatgg aagcttacag aaaacaaatg 1500 ctatataaca tagaacttat ggtaaatgcc gacaacgcga tagattatgc ccacgcaaag 1560 ttggccccat tgccatttga gtcatgtttg gttgatgact gtataaagag aggaatgtcc 1620 gctcaggaag gcggcgcaat ctataatttc actggtccac agggctttgg tattgcaaac 1680 gttgctgata gcttgtatac gattaagaaa ttggtgttcg aggagaagag aattacgatg 1740 ggtgaattaa agaaagcgtt ggaaatgaat tatggtaagg gtttggatgc cacaaccgct 1800 ggtgacatcg caatgcaggt cgcgaaggga ctaaaagatg ccggacagga agtgggtccc 1860 gacgtgatcg ctaatacaat ccgtcaagtt cttgaaatgg aattaccaga agatgtaaga 1920 aagagatatg aagagatcca tgaaatgata cttgagttac caaagtatgg taatgatata 1980 gatgaagttg atgaattagc tagagaagca gcttactttt acacaagacc attagaaact 2040 tttaagaatc caaggggtgg catgtatcaa gccggccttt atcccgtgtc cgctaatgtg 2100 ccactaggcg ctcaaacggg ggccacaccc gatggacgtt tggcgcatac acccgtggcg 2160 gatggcgttg gtccgacatc aggcttcgat atatccggac caacagcttc ttgcaattct 2220 gtcgccaagt tggatcatgc tatagcctct aatggtacct tatttaatat gaagatgcac 2280 ccaaccgcaa tggcaggtga aaagggctta gaatccttca tatcgttgat ccgtggttat 2340 ttcgatcaac aaggtatgca catgcaattt aacgtagtag acagggctac actgcttgat 2400 gcgcaggccc accctgaaaa gtattcaggc ttaattgtca gagtggcagg ttattctgcc 2460 ctttttacca cattgtccaa gtcattacaa gatgatataa tcaaacgtac cgaacaagca 2520 gacaatagat ag 2532

<210> SEQ ID NO 16 <211> LENGTH: 843 <212> TYPE: PRT <213> ORGANISM: Roseburia inulinivorans <400> SEQUENCE: 16 Met Gly Asn Tyr Asp Ser Thr Pro Ile Ala Lys Ser Asp Arg Ile Lys 1 5 10 15 Arg Leu Val Asp His Leu Tyr Ala Lys Met Pro Glu Ile Glu Ala Ala 20 25 30 Arg Ala Glu Leu Ile Thr Glu Ser Phe Lys Ala Thr Glu Gly Gln Pro 35 40 45 Val Val Met Arg Lys Ala Arg Ala Phe Glu His Ile Leu Lys Asn Leu 50 55 60 Pro Ile Ile Ile Arg Pro Glu Glu Leu Ile Val Gly Ser Thr Thr Ile 65 70 75 80 Ala Pro Arg Gly Cys Gln Thr Tyr Pro Glu Phe Ser Tyr Glu Trp Leu 85 90 95 Glu Ala Glu Phe Glu Thr Val Glu Thr Arg Ser Ala Asp Pro Phe Tyr 100 105 110 Ile Ser Glu Glu Thr Lys Lys Arg Leu Leu Ala Ala Asp Ala Tyr Trp 115 120 125 Lys Gly Lys Thr Thr Ser Glu Leu Ala Thr Ser Tyr Met Ala Pro Glu 130 135 140 Thr Leu Arg Ala Met Lys His Asn Phe Phe Thr Pro Gly Asn Tyr Phe 145 150 155 160 Tyr Asn Gly Val Gly His Val Thr Val Gln Tyr Glu Thr Val Leu Ala 165 170 175 Ile Gly Leu Asn Gly Val Lys Glu Lys Val Arg Lys Glu Met Glu Asn 180 185 190 Cys His Phe Gly Asp Ala Asp Tyr Ser Thr Lys Met Cys Phe Leu Glu 195 200 205 Ser Ile Leu Ile Ser Cys Asp Ala Val Ile Thr Tyr Ala Asn Arg Tyr 210 215 220 Ala Lys Met Ala Glu Glu Met Ala Glu Lys Glu Thr Asp Ala Ala Arg 225 230 235 240 Arg Gln Glu Leu Leu Thr Ile Ala Arg Val Cys Lys Asn Val Pro Glu 245 250 255 Phe Pro Ala Glu Ser Phe Gln Glu Ala Cys Gln Ser Phe Trp Phe Ile 260 265 270 Gln Gln Val Leu Gln Ile Glu Ser Ser Gly His Ser Ile Ser Pro Gly 275 280 285 Arg Phe Asp Gln Tyr Met Tyr Pro Tyr Tyr Glu Lys Asp Leu Lys Glu 290 295 300 Gly Ser Leu Thr Arg Glu Tyr Ala Gln Glu Leu Ile Asp Cys Ile Trp 305 310 315 320 Val Lys Leu Asn Asp Leu Asn Lys Cys Arg Asp Ala Ala Ser Ala Glu 325 330 335 Gly Phe Ala Gly Tyr Ser Leu Phe Gln Asn Leu Ile Val Gly Gly Gln 340 345 350 Thr Val Gln Gly Arg Asp Ala Thr Asn Asp Leu Ser Phe Met Cys Ile 355 360 365 Thr Ala Ser Glu His Val Phe Leu Pro Met Pro Ser Leu Ser Ile Arg 370 375 380 Val Trp His Gly Ser Ser Lys Ala Leu Leu Met Arg Ala Ala Glu Leu 385 390 395 400 Thr Arg Thr Gly Ile Gly Leu Pro Ala Tyr Tyr Asn Asp Glu Val Ile 405 410 415 Ile Pro Ala Leu Val His Arg Gly Ala Thr Met Asp Glu Ala Arg Asn 420 425 430 Tyr Asn Ile Ile Gly Cys Val Glu Pro Gln Val Pro Gly Lys Thr Asp 435 440 445 Gly Trp His Asp Ala Ala Phe Phe Asn Met Cys Arg Pro Leu Glu Met 450 455 460 Val Phe Ser Asn Gly Tyr Asp Asn Gly Glu Ile Ala Ser Ile Gln Thr 465 470 475 480 Gly Asn Val Glu Ser Phe Gln Ser Phe Asp Glu Phe Met Glu Ala Tyr 485 490 495 Arg Lys Gln Met Leu Tyr Asn Ile Glu Leu Met Val Asn Ala Asp Asn 500 505 510 Ala Ile Asp Tyr Ala His Ala Lys Leu Ala Pro Leu Pro Phe Glu Ser 515 520 525 Cys Leu Val Asp Asp Cys Ile Lys Arg Gly Met Ser Ala Gln Glu Gly 530 535 540 Gly Ala Ile Tyr Asn Phe Thr Gly Pro Gln Gly Phe Gly Ile Ala Asn 545 550 555 560 Val Ala Asp Ser Leu Tyr Thr Ile Lys Lys Leu Val Phe Glu Glu Lys 565 570 575 Arg Ile Thr Met Gly Glu Leu Lys Lys Ala Leu Glu Met Asn Tyr Gly 580 585 590 Lys Gly Leu Asp Ala Thr Thr Ala Gly Asp Ile Ala Met Gln Val Ala 595 600 605 Lys Gly Leu Lys Asp Ala Gly Gln Glu Val Gly Pro Asp Val Ile Ala 610 615 620 Asn Thr Ile Arg Gln Val Leu Glu Met Glu Leu Pro Glu Asp Val Arg 625 630 635 640 Lys Arg Tyr Glu Glu Ile His Glu Met Ile Leu Glu Leu Pro Lys Tyr 645 650 655 Gly Asn Asp Ile Asp Glu Val Asp Glu Leu Ala Arg Glu Ala Ala Tyr 660 665 670 Phe Tyr Thr Arg Pro Leu Glu Thr Phe Lys Asn Pro Arg Gly Gly Met 675 680 685 Tyr Gln Ala Gly Leu Tyr Pro Val Ser Ala Asn Val Pro Leu Gly Ala 690 695 700 Gln Thr Gly Ala Thr Pro Asp Gly Arg Leu Ala His Thr Pro Val Ala 705 710 715 720 Asp Gly Val Gly Pro Thr Ser Gly Phe Asp Ile Ser Gly Pro Thr Ala 725 730 735 Ser Cys Asn Ser Val Ala Lys Leu Asp His Ala Ile Ala Ser Asn Gly 740 745 750 Thr Leu Phe Asn Met Lys Met His Pro Thr Ala Met Ala Gly Glu Lys 755 760 765 Gly Leu Glu Ser Phe Ile Ser Leu Ile Arg Gly Tyr Phe Asp Gln Gln 770 775 780 Gly Met His Met Gln Phe Asn Val Val Asp Arg Ala Thr Leu Leu Asp 785 790 795 800 Ala Gln Ala His Pro Glu Lys Tyr Ser Gly Leu Ile Val Arg Val Ala 805 810 815 Gly Tyr Ser Ala Leu Phe Thr Thr Leu Ser Lys Ser Leu Gln Asp Asp 820 825 830 Ile Ile Lys Arg Thr Glu Gln Ala Asp Asn Arg 835 840 <210> SEQ ID NO 17 <211> LENGTH: 794 <212> TYPE: DNA <213> ORGANISM: Roseburia inulinivorans <400> SEQUENCE: 17 atgaaagaat atcttaatac ttcaggtaga atatttgata tccagaggta ttctattcac 60 gatggccctg gtgtgcgtac aattgtgttt ctaaaaggtt gtgcccttag atgcagatgg 120 tgctgtaatc ctgaaagcca aagcttcgaa gttgaaacaa tgacgattaa tggaaaacct 180 aaagtcatgg gtaaagatgt tacagtcgcc gaggttatga agacggtaga aagagacatg 240 ccttattacc ttcaatcagg tggtggtatc accttatcgg gtggcgaatg tactttgcaa 300 ccagaatttt cccttggcct attgagagct gcaaaggatt tgggcatatc cacggcaata 360 gagagcatgg cgtacgcaaa gtacgaagta atagaaactc ttcttccgta tttggatacg 420 tatttaatgg acatcaaaca tatgaatcct gagaaacata aagaatacac tggtcatgat 480 aacttgagga tgttagaaaa cgccttaaga gtcgcgcatt ctggtcagac cgaactgatc 540 atcagagtac ctgtcatccc aggattcaac gcaactgagc aggaactact agatattgca 600 aaattcgcag atacactgcc tggagttaga caaatacaca tcttgccata tcataatttt 660 ggtcagggta aatacgaagg attgaacagg gactatccga tgggggacac tgagaaaccc 720 tctaatgaac agatgaaagc ttttcaagaa atgattcaaa agaacacttc cctacattgc 780 caaatcggtg gtta 794 <210> SEQ ID NO 18 <211> LENGTH: 264 <212> TYPE: PRT <213> ORGANISM: Roseburia inulinivorans <400> SEQUENCE: 18 Met Lys Glu Tyr Leu Asn Thr Ser Gly Arg Ile Phe Asp Ile Gln Arg 1 5 10 15 Tyr Ser Ile His Asp Gly Pro Gly Val Arg Thr Ile Val Phe Leu Lys 20 25 30 Gly Cys Ala Leu Arg Cys Arg Trp Cys Cys Asn Pro Glu Ser Gln Ser 35 40 45 Phe Glu Val Glu Thr Met Thr Ile Asn Gly Lys Pro Lys Val Met Gly 50 55 60 Lys Asp Val Thr Val Ala Glu Val Met Lys Thr Val Glu Arg Asp Met 65 70 75 80 Pro Tyr Tyr Leu Gln Ser Gly Gly Gly Ile Thr Leu Ser Gly Gly Glu 85 90 95 Cys Thr Leu Gln Pro Glu Phe Ser Leu Gly Leu Leu Arg Ala Ala Lys 100 105 110 Asp Leu Gly Ile Ser Thr Ala Ile Glu Ser Met Ala Tyr Ala Lys Tyr 115 120 125 Glu Val Ile Glu Thr Leu Leu Pro Tyr Leu Asp Thr Tyr Leu Met Asp 130 135 140 Ile Lys His Met Asn Pro Glu Lys His Lys Glu Tyr Thr Gly His Asp 145 150 155 160 Asn Leu Arg Met Leu Glu Asn Ala Leu Arg Val Ala His Ser Gly Gln 165 170 175 Thr Glu Leu Ile Ile Arg Val Pro Val Ile Pro Gly Phe Asn Ala Thr 180 185 190 Glu Gln Glu Leu Leu Asp Ile Ala Lys Phe Ala Asp Thr Leu Pro Gly 195 200 205 Val Arg Gln Ile His Ile Leu Pro Tyr His Asn Phe Gly Gln Gly Lys

210 215 220 Tyr Glu Gly Leu Asn Arg Asp Tyr Pro Met Gly Asp Thr Glu Lys Pro 225 230 235 240 Ser Asn Glu Gln Met Lys Ala Phe Gln Glu Met Ile Gln Lys Asn Thr 245 250 255 Ser Leu His Cys Gln Ile Gly Gly 260 <210> SEQ ID NO 19 <211> LENGTH: 978 <212> TYPE: DNA <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 19 atggctgata aacaacgtaa aaaagttatc cttgtaggtg acggtgctgt aggttcatca 60 tacgcttttg ctcttgtaaa ccaagggatt gcacaagaat taggaattgt tgaccttttt 120 aaagaaaaaa ctcaaggaga tgcagaagac ctttctcatg ccttggcatt tacttcacct 180 aaaaagattt actctgcaga ctactctgat gcaagcgacg ctgacctcgt agtcttgact 240 tctggtgctc cacaaaaacc aggtgaaact cgtcttgacc ttgttgaaaa aaatcttcgt 300 atcactaaag atgttgtcac taaaattgtt gcttcaggtt tcaaaggaat cttccttgtt 360 gctgctaacc cagttgatat cttgacatac gctacttgga aattctcagg tttccctaaa 420 aaccgcgttg taggttcagg tacttcactt gatactgcac gtttccgtca agcattggca 480 gaaaaagttg atgttgacgc tcgttcaatc cacgcataca tcatgggtga acacggtgac 540 tcagaatttg ccgtttggtc acacgctaac gttgctggtg ttaaattgga acaatggttc 600 caagaaaatg actaccttaa cgaagctgaa atcgttgaat tgtttgaatc tgtacgtgat 660 gctgcttact caatcatcgc taaaaaaggt gcaacattct atggtgtcgc tgtagctctt 720 gctcgtatta ctaaagcaat tcttgatgat gaacatgcag tacttccagt atcagtattc 780 caagatggac aatatggcgt aagcgactgc taccttggtc aaccagctgt agttggtgct 840 gaaggtgttg ttaacccaat ccacattcca ttgaatgatg ctgaaatgca aaaaatggaa 900 gcttctggtg ctcaattgaa agcaatcatt gacgaagctt ttgctaaaga agaatttgct 960 tctgcagtta aaaactaa 978 <210> SEQ ID NO 20 <211> LENGTH: 325 <212> TYPE: PRT <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 20 Met Ala Asp Lys Gln Arg Lys Lys Val Ile Leu Val Gly Asp Gly Ala 1 5 10 15 Val Gly Ser Ser Tyr Ala Phe Ala Leu Val Asn Gln Gly Ile Ala Gln 20 25 30 Glu Leu Gly Ile Val Asp Leu Phe Lys Glu Lys Thr Gln Gly Asp Ala 35 40 45 Glu Asp Leu Ser His Ala Leu Ala Phe Thr Ser Pro Lys Lys Ile Tyr 50 55 60 Ser Ala Asp Tyr Ser Asp Ala Ser Asp Ala Asp Leu Val Val Leu Thr 65 70 75 80 Ser Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Asp Leu Val Glu 85 90 95 Lys Asn Leu Arg Ile Thr Lys Asp Val Val Thr Lys Ile Val Ala Ser 100 105 110 Gly Phe Lys Gly Ile Phe Leu Val Ala Ala Asn Pro Val Asp Ile Leu 115 120 125 Thr Tyr Ala Thr Trp Lys Phe Ser Gly Phe Pro Lys Asn Arg Val Val 130 135 140 Gly Ser Gly Thr Ser Leu Asp Thr Ala Arg Phe Arg Gln Ala Leu Ala 145 150 155 160 Glu Lys Val Asp Val Asp Ala Arg Ser Ile His Ala Tyr Ile Met Gly 165 170 175 Glu His Gly Asp Ser Glu Phe Ala Val Trp Ser His Ala Asn Val Ala 180 185 190 Gly Val Lys Leu Glu Gln Trp Phe Gln Glu Asn Asp Tyr Leu Asn Glu 195 200 205 Ala Glu Ile Val Glu Leu Phe Glu Ser Val Arg Asp Ala Ala Tyr Ser 210 215 220 Ile Ile Ala Lys Lys Gly Ala Thr Phe Tyr Gly Val Ala Val Ala Leu 225 230 235 240 Ala Arg Ile Thr Lys Ala Ile Leu Asp Asp Glu His Ala Val Leu Pro 245 250 255 Val Ser Val Phe Gln Asp Gly Gln Tyr Gly Val Ser Asp Cys Tyr Leu 260 265 270 Gly Gln Pro Ala Val Val Gly Ala Glu Gly Val Val Asn Pro Ile His 275 280 285 Ile Pro Leu Asn Asp Ala Glu Met Gln Lys Met Glu Ala Ser Gly Ala 290 295 300 Gln Leu Lys Ala Ile Ile Asp Glu Ala Phe Ala Lys Glu Glu Phe Ala 305 310 315 320 Ser Ala Val Lys Asn 325 <210> SEQ ID NO 21 <211> LENGTH: 996 <212> TYPE: DNA <213> ORGANISM: Leuconostoc mesenteroides <400> SEQUENCE: 21 atgaagattt ttgcttacgg cattcgtgat gatgaaaagc catcacttga agaatggaaa 60 gcggctaacc cagagattga agtggactac acacaagaat tattgacacc tgaaacagct 120 aagttggctg agggatcaga ttcagctgtt gtttatcaac aattggacta tacacgtgaa 180 acattgacag ctttagctaa cgttggtgtt actaacttgt cattgcgtaa cgttggtaca 240 gataacattg attttgatgc agcacgtgaa tttaacttta acatttcaaa tgttcctgtt 300 tattcaccaa atgctattgc agaacactca atgattcaat tatctcgttt gctacgtcgc 360 acgaaagcat tggatgccaa aattgctaag cacgacttgc gttgggcacc aacaattgga 420 cgtgaaatgc gtatgcaaac agttggtgtt attggtacag gtcatattgg ccgtgttgct 480 attaacattt tgaaaggctt tggggccaag gttattgctt atgacaagta cccaaatgct 540 gaattacaag cagaaggttt gtacgttgac acattagacg aattatatgc acaagctgat 600 gcaatttcat tgtatgttcc tggtgtacct gaaaaccatc atctaatcaa tgcagatgct 660 attgctaaga tgaaggatgg tgtggttatc atgaacgctg cgcgtggtaa tttgatggac 720 attgacgcta ttattgatgg tttgaattct ggtaagattt cagacttcgg tatggacgtt 780 tatgaaaatg aagttggctt gttcaatgaa gattggtctg gtaaagaatt cccagatgct 840 aagattgctg acttgattgc acgcgaaaat gtattggtta cgccacacac ggctttctat 900 acaactaaag ctgttctaga aatggttcac caatcatttg atgcagcagt tgctttcgcc 960 aagggtgaga agccagctat tgctgttgaa tattaa 996 <210> SEQ ID NO 22 <211> LENGTH: 331 <212> TYPE: PRT <213> ORGANISM: Leuconostoc mesenteroides <400> SEQUENCE: 22 Met Lys Ile Phe Ala Tyr Gly Ile Arg Asp Asp Glu Lys Pro Ser Leu 1 5 10 15 Glu Glu Trp Lys Ala Ala Asn Pro Glu Ile Glu Val Asp Tyr Thr Gln 20 25 30 Glu Leu Leu Thr Pro Glu Thr Ala Lys Leu Ala Glu Gly Ser Asp Ser 35 40 45 Ala Val Val Tyr Gln Gln Leu Asp Tyr Thr Arg Glu Thr Leu Thr Ala 50 55 60 Leu Ala Asn Val Gly Val Thr Asn Leu Ser Leu Arg Asn Val Gly Thr 65 70 75 80 Asp Asn Ile Asp Phe Asp Ala Ala Arg Glu Phe Asn Phe Asn Ile Ser 85 90 95 Asn Val Pro Val Tyr Ser Pro Asn Ala Ile Ala Glu His Ser Met Ile 100 105 110 Gln Leu Ser Arg Leu Leu Arg Arg Thr Lys Ala Leu Asp Ala Lys Ile 115 120 125 Ala Lys His Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Met Arg 130 135 140 Met Gln Thr Val Gly Val Ile Gly Thr Gly His Ile Gly Arg Val Ala 145 150 155 160 Ile Asn Ile Leu Lys Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp Lys 165 170 175 Tyr Pro Asn Ala Glu Leu Gln Ala Glu Gly Leu Tyr Val Asp Thr Leu 180 185 190 Asp Glu Leu Tyr Ala Gln Ala Asp Ala Ile Ser Leu Tyr Val Pro Gly 195 200 205 Val Pro Glu Asn His His Leu Ile Asn Ala Asp Ala Ile Ala Lys Met 210 215 220 Lys Asp Gly Val Val Ile Met Asn Ala Ala Arg Gly Asn Leu Met Asp 225 230 235 240 Ile Asp Ala Ile Ile Asp Gly Leu Asn Ser Gly Lys Ile Ser Asp Phe 245 250 255 Gly Met Asp Val Tyr Glu Asn Glu Val Gly Leu Phe Asn Glu Asp Trp 260 265 270 Ser Gly Lys Glu Phe Pro Asp Ala Lys Ile Ala Asp Leu Ile Ala Arg 275 280 285 Glu Asn Val Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr Lys Ala 290 295 300 Val Leu Glu Met Val His Gln Ser Phe Asp Ala Ala Val Ala Phe Ala 305 310 315 320 Lys Gly Glu Lys Pro Ala Ile Ala Val Glu Tyr 325 330 <210> SEQ ID NO 23 <211> LENGTH: 987 <212> TYPE: DNA <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 23 atgactgcaa ctaaactaca caaaaaagtc atccttgttg gtgacggtgc cgtaggttca 60 tcttacgctt tcgcacttgt aaaccaaggt atcgctcaag aactaggtat catcgaaatt 120 ccacaattat ttgaaaaagc cgttggtgat gcgcttgacc ttagccacgc acttcctttc 180

acttcaccta aaaaaatcta tgcagctaaa tatgaagact gtgcggatgc tgaccttgta 240 gttatcactg ctggtgctcc tcaaaaacca ggtgagactc gtcttgatct tgttggtaaa 300 aaccttgcaa tcaacaaatc aatcgttact caagttgttg aatcaggatt caacggtatt 360 ttccttgtag ctgctaaccc agtagacgta ttgacttact ctacatggaa gttctcagga 420 ttccctaaag aacgcgttat cggttcaggt acttcacttg actcagctcg tttccgtcaa 480 gcacttgctg aaaaacttaa tgtcgatgct cgttcagttc acgcttacat catgggtgaa 540 cacggcgact cagagtttgc ggtttggtca cacgctaaca tcgccggtgt aaaccttgaa 600 gagttcctta aagacgaaga aaacgttcaa gaagctgaat tagttgaatt gttcgaaggt 660 gttcgtgatg cagcttacac aattatcaac aaaaaaggtg ctacatacta cggtatcgca 720 gtagcccttg ctcgtatcac taaagctatc cttgacgatg aaaatgcagt acttccattg 780 tctgtattcc aagaaggtca atatggtgta aacaacatct ttatcggtca acctgctatt 840 gtaggcgcac acggtatcgt acgtccagta aacatcccat tgaacgatgc tgaacaacaa 900 aagatgaagg cttctgccga tgaattgcaa gctatcattg atgaagcatg gaaaaaccct 960 gaattccaag aagcttcaaa aaactaa 987 <210> SEQ ID NO 24 <211> LENGTH: 328 <212> TYPE: PRT <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 24 Met Thr Ala Thr Lys Leu His Lys Lys Val Ile Leu Val Gly Asp Gly 1 5 10 15 Ala Val Gly Ser Ser Tyr Ala Phe Ala Leu Val Asn Gln Gly Ile Ala 20 25 30 Gln Glu Leu Gly Ile Ile Glu Ile Pro Gln Leu Phe Glu Lys Ala Val 35 40 45 Gly Asp Ala Leu Asp Leu Ser His Ala Leu Pro Phe Thr Ser Pro Lys 50 55 60 Lys Ile Tyr Ala Ala Lys Tyr Glu Asp Cys Ala Asp Ala Asp Leu Val 65 70 75 80 Val Ile Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Asp 85 90 95 Leu Val Gly Lys Asn Leu Ala Ile Asn Lys Ser Ile Val Thr Gln Val 100 105 110 Val Glu Ser Gly Phe Asn Gly Ile Phe Leu Val Ala Ala Asn Pro Val 115 120 125 Asp Val Leu Thr Tyr Ser Thr Trp Lys Phe Ser Gly Phe Pro Lys Glu 130 135 140 Arg Val Ile Gly Ser Gly Thr Ser Leu Asp Ser Ala Arg Phe Arg Gln 145 150 155 160 Ala Leu Ala Glu Lys Leu Asn Val Asp Ala Arg Ser Val His Ala Tyr 165 170 175 Ile Met Gly Glu His Gly Asp Ser Glu Phe Ala Val Trp Ser His Ala 180 185 190 Asn Ile Ala Gly Val Asn Leu Glu Glu Phe Leu Lys Asp Glu Glu Asn 195 200 205 Val Gln Glu Ala Glu Leu Val Glu Leu Phe Glu Gly Val Arg Asp Ala 210 215 220 Ala Tyr Thr Ile Ile Asn Lys Lys Gly Ala Thr Tyr Tyr Gly Ile Ala 225 230 235 240 Val Ala Leu Ala Arg Ile Thr Lys Ala Ile Leu Asp Asp Glu Asn Ala 245 250 255 Val Leu Pro Leu Ser Val Phe Gln Glu Gly Gln Tyr Gly Val Asn Asn 260 265 270 Ile Phe Ile Gly Gln Pro Ala Ile Val Gly Ala His Gly Ile Val Arg 275 280 285 Pro Val Asn Ile Pro Leu Asn Asp Ala Glu Gln Gln Lys Met Lys Ala 290 295 300 Ser Ala Asp Glu Leu Gln Ala Ile Ile Asp Glu Ala Trp Lys Asn Pro 305 310 315 320 Glu Phe Gln Glu Ala Ser Lys Asn 325 <210> SEQ ID NO 25 <211> LENGTH: 996 <212> TYPE: DNA <213> ORGANISM: Pediococcus pentosaceus <400> SEQUENCE: 25 atgaaaatta ttgcttatgg cattcgagat gacgaaaaac cttacctaga agaatgggtt 60 aaagataata aaattgaagt aaaggctgtt agcgaattgt tggactccaa cacgattgaa 120 caagctaagg gttatgacgg agttgttgca tatcaacaga aaccttatac agatgatttg 180 ttcgataaaa tgaatgaatt cgggattcat gccttttcgc ttcgtaacgt tggtgttgat 240 aatgttccag ttgaggcttt aaagcgaaat aatattaaga ttaccaatgt tccagcgtac 300 tctccaatgg cgattgcaga actttcagta acccaactcc tagctttaat tcgtcgaatt 360 ccagaatttg atgctaagat ggctcgtggt gatttcagat gggaaccaga tattgctcta 420 gaacttaacc aaatgacagt aggagttatt ggtaccggaa gaattgggcg tgcggccatt 480 aatatcttta aaggctttgg agctaaagtg attgcttatg atgttttccg aaattcagaa 540 cttgaaaaag aaggaatcta tgttgactcg cttgaagaac tttatcgtca agtagatgtt 600 attaccttac atgttcccgc tttaaaagat aactaccata tgttaaatga tgaagcgttc 660 gcacagatgc atgatggggt atttgttcta aattttgctc gcggtagctt gattgacacg 720 aaggcattac ttaaggcttt agatagtggt aaggtggctg gtgcggcact agatacctat 780 gaagacgaag taggtatttt tgatgtggat caccaaaatg acccaatcaa tgatcccgta 840 tttaatgatt tatacagtag acgtaatgta aaaatcacac cacatgcggc tttttatact 900 aagccagcag ttaaaaatat ggtacaaatt gctcttgaaa ataataaagc actaattgaa 960 aaaggtgctg caagaaatga agttaagttt gactaa 996 <210> SEQ ID NO 26 <211> LENGTH: 331 <212> TYPE: PRT <213> ORGANISM: Pediococcus pentosaceus <400> SEQUENCE: 26 Met Lys Ile Ile Ala Tyr Gly Ile Arg Asp Asp Glu Lys Pro Tyr Leu 1 5 10 15 Glu Glu Trp Val Lys Asp Asn Lys Ile Glu Val Lys Ala Val Ser Glu 20 25 30 Leu Leu Asp Ser Asn Thr Ile Glu Gln Ala Lys Gly Tyr Asp Gly Val 35 40 45 Val Ala Tyr Gln Gln Lys Pro Tyr Thr Asp Asp Leu Phe Asp Lys Met 50 55 60 Asn Glu Phe Gly Ile His Ala Phe Ser Leu Arg Asn Val Gly Val Asp 65 70 75 80 Asn Val Pro Val Glu Ala Leu Lys Arg Asn Asn Ile Lys Ile Thr Asn 85 90 95 Val Pro Ala Tyr Ser Pro Met Ala Ile Ala Glu Leu Ser Val Thr Gln 100 105 110 Leu Leu Ala Leu Ile Arg Arg Ile Pro Glu Phe Asp Ala Lys Met Ala 115 120 125 Arg Gly Asp Phe Arg Trp Glu Pro Asp Ile Ala Leu Glu Leu Asn Gln 130 135 140 Met Thr Val Gly Val Ile Gly Thr Gly Arg Ile Gly Arg Ala Ala Ile 145 150 155 160 Asn Ile Phe Lys Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp Val Phe 165 170 175 Arg Asn Ser Glu Leu Glu Lys Glu Gly Ile Tyr Val Asp Ser Leu Glu 180 185 190 Glu Leu Tyr Arg Gln Val Asp Val Ile Thr Leu His Val Pro Ala Leu 195 200 205 Lys Asp Asn Tyr His Met Leu Asn Asp Glu Ala Phe Ala Gln Met His 210 215 220 Asp Gly Val Phe Val Leu Asn Phe Ala Arg Gly Ser Leu Ile Asp Thr 225 230 235 240 Lys Ala Leu Leu Lys Ala Leu Asp Ser Gly Lys Val Ala Gly Ala Ala 245 250 255 Leu Asp Thr Tyr Glu Asp Glu Val Gly Ile Phe Asp Val Asp His Gln 260 265 270 Asn Asp Pro Ile Asn Asp Pro Val Phe Asn Asp Leu Tyr Ser Arg Arg 275 280 285 Asn Val Lys Ile Thr Pro His Ala Ala Phe Tyr Thr Lys Pro Ala Val 290 295 300 Lys Asn Met Val Gln Ile Ala Leu Glu Asn Asn Lys Ala Leu Ile Glu 305 310 315 320 Lys Gly Ala Ala Arg Asn Glu Val Lys Phe Asp 325 330 <210> SEQ ID NO 27 <211> LENGTH: 1110 <212> TYPE: DNA <213> ORGANISM: Pediococcus pentosaceus <400> SEQUENCE: 27 atgacaatga ttaatggtta tgaacaaagt gatcgtgaag aaaaaattga cattttaaat 60 ttggagtctt tggaagaaag agccgaaaag attattccaa ctggtgggtt tggatatatc 120 tctggtggtt ctgaagatga atggactctc cgacaaaatc gaactgcatt ccagcatcga 180 caaatcgcgc ccaaagcttt gagtggaatt gaaaaaccag aactaaatac agaaatcttt 240 ggaattccat tgaatactcc agtgatgatg gcgccagctg cagctcaagg cttagcacat 300 tcacaaggtg aaaaagatac agctagaggt cttgccgcag taggaggctt aatggcacaa 360 agcacatatt catcagtttc tattgctgat acggcagctg ctggtgaagg tgctcctcaa 420 tttttccagc tttacatgag taaggactgg aattttaatg agagcttgct agatgaggct 480 aaaaaagctc atgttaaagc aattattttg accgtagatg ccactgttga tggttatcga 540 gaagctgata ttaaaaataa gtttgcattt ccacttccaa tggctaactt aactaagttt 600 tccgagggtg atggtcaagg aaaaggaatt gaagaaatct acgcttctgc agctcaaaat 660 ataagaccgg aagatgttag aagaattgct gattacacac aattacccgt aattgttaaa 720 ggaattcaaa ctcctgagga tgctattcga gcaattgatg ctggggcagc cggcatttat 780 gtatcaaacc atggaggtcg tcagctaaac gggggacctg gatcttttga tgttttggaa 840 gatatcgcta cctccgttaa taagcaggtg ccaattatct ttgatagtgg tgtacgtcgt 900 ggttcagatg tatttaaagc tttggctagt ggcgcagaca tcgtggcttt gggtcgtcca 960

gtaatttatg gattagcttt aggtggtgcc aaaggggttc aatctgtatt tgaacatata 1020 gaccatgaac ttgaaattgt gatgcaacta gcaggtacta aaaccattga tgatattaaa 1080 aataacccac tactaaacat caaatattaa 1110 <210> SEQ ID NO 28 <211> LENGTH: 369 <212> TYPE: PRT <213> ORGANISM: Pediococcus pentosaceus <400> SEQUENCE: 28 Met Thr Met Ile Asn Gly Tyr Glu Gln Ser Asp Arg Glu Glu Lys Ile 1 5 10 15 Asp Ile Leu Asn Leu Glu Ser Leu Glu Glu Arg Ala Glu Lys Ile Ile 20 25 30 Pro Thr Gly Gly Phe Gly Tyr Ile Ser Gly Gly Ser Glu Asp Glu Trp 35 40 45 Thr Leu Arg Gln Asn Arg Thr Ala Phe Gln His Arg Gln Ile Ala Pro 50 55 60 Lys Ala Leu Ser Gly Ile Glu Lys Pro Glu Leu Asn Thr Glu Ile Phe 65 70 75 80 Gly Ile Pro Leu Asn Thr Pro Val Met Met Ala Pro Ala Ala Ala Gln 85 90 95 Gly Leu Ala His Ser Gln Gly Glu Lys Asp Thr Ala Arg Gly Leu Ala 100 105 110 Ala Val Gly Gly Leu Met Ala Gln Ser Thr Tyr Ser Ser Val Ser Ile 115 120 125 Ala Asp Thr Ala Ala Ala Gly Glu Gly Ala Pro Gln Phe Phe Gln Leu 130 135 140 Tyr Met Ser Lys Asp Trp Asn Phe Asn Glu Ser Leu Leu Asp Glu Ala 145 150 155 160 Lys Lys Ala His Val Lys Ala Ile Ile Leu Thr Val Asp Ala Thr Val 165 170 175 Asp Gly Tyr Arg Glu Ala Asp Ile Lys Asn Lys Phe Ala Phe Pro Leu 180 185 190 Pro Met Ala Asn Leu Thr Lys Phe Ser Glu Gly Asp Gly Gln Gly Lys 195 200 205 Gly Ile Glu Glu Ile Tyr Ala Ser Ala Ala Gln Asn Ile Arg Pro Glu 210 215 220 Asp Val Arg Arg Ile Ala Asp Tyr Thr Gln Leu Pro Val Ile Val Lys 225 230 235 240 Gly Ile Gln Thr Pro Glu Asp Ala Ile Arg Ala Ile Asp Ala Gly Ala 245 250 255 Ala Gly Ile Tyr Val Ser Asn His Gly Gly Arg Gln Leu Asn Gly Gly 260 265 270 Pro Gly Ser Phe Asp Val Leu Glu Asp Ile Ala Thr Ser Val Asn Lys 275 280 285 Gln Val Pro Ile Ile Phe Asp Ser Gly Val Arg Arg Gly Ser Asp Val 290 295 300 Phe Lys Ala Leu Ala Ser Gly Ala Asp Ile Val Ala Leu Gly Arg Pro 305 310 315 320 Val Ile Tyr Gly Leu Ala Leu Gly Gly Ala Lys Gly Val Gln Ser Val 325 330 335 Phe Glu His Ile Asp His Glu Leu Glu Ile Val Met Gln Leu Ala Gly 340 345 350 Thr Lys Thr Ile Asp Asp Ile Lys Asn Asn Pro Leu Leu Asn Ile Lys 355 360 365 Tyr <210> SEQ ID NO 29 <211> LENGTH: 972 <212> TYPE: DNA <213> ORGANISM: Lactobacillus acidophilus <400> SEQUENCE: 29 atggcaagag ttgaaaaacc tcgtaaagtt attttagttg gtgacggtgc tgtaggttct 60 acctttgcat tttcaatggt gcaacaaggt attgctgaag aattaggtat cattgatatt 120 gctaaggaac acgttgaagg tgacgcaatc gacttagcag atgctactcc atggactttc 180 ccaaagaaca tttacgcagc tgactacgct gactgcaagg acgcagactt agtagttatt 240 actgctggtg ctccacaaaa gccaggtgaa actcgtcttg accttgttaa caagaacttg 300 aagattttat catcaatcgt tgaaccagtt gttgaatcag gctttgaagg tatcttctta 360 gtagttgcta acccagttga catcttgact cacgcaactt ggaagatttc aggcttccct 420 aaggatcgcg ttattggttc aggtacttca cttgatactg gtcgtcttca aaaggttatc 480 ggtaagatgg aacacgttga cccacgttca gttaatgcat acatgcttgg tgaacacggt 540 gatactgaat tcccagtatg gagctacaac aatgttggtg gcgtaaaggt tagcgactgg 600 gttaaggctc acggtatgga tgaatctaag cttgaagaaa tccacaagga agttgctgac 660 atggcttacg acattatcaa caagaagggt gctactttct acggtatcgg tacagcttca 720 gcaatgatcg ctaaggctat cttgaacgat gaacaccgtg tacttccact ctcagttgca 780 atggatggtc aatacggttt acacgacctt cacattggta ctcctgcagt tgttggccgt 840 aacggtcttg aacaaattat tgaaatgcct ttaaccgctg atgaacaagc taagatggaa 900 gcttctgcta agcaattaaa ggaagttatg gacaaagcct ttgaagaaac tggcgttaag 960 gttcgtcaat aa 972 <210> SEQ ID NO 30 <211> LENGTH: 323 <212> TYPE: PRT <213> ORGANISM: Lactobacillus acidophilus <400> SEQUENCE: 30 Met Ala Arg Val Glu Lys Pro Arg Lys Val Ile Leu Val Gly Asp Gly 1 5 10 15 Ala Val Gly Ser Thr Phe Ala Phe Ser Met Val Gln Gln Gly Ile Ala 20 25 30 Glu Glu Leu Gly Ile Ile Asp Ile Ala Lys Glu His Val Glu Gly Asp 35 40 45 Ala Ile Asp Leu Ala Asp Ala Thr Pro Trp Thr Phe Pro Lys Asn Ile 50 55 60 Tyr Ala Ala Asp Tyr Ala Asp Cys Lys Asp Ala Asp Leu Val Val Ile 65 70 75 80 Thr Ala Gly Ala Pro Gln Lys Pro Gly Glu Thr Arg Leu Asp Leu Val 85 90 95 Asn Lys Asn Leu Lys Ile Leu Ser Ser Ile Val Glu Pro Val Val Glu 100 105 110 Ser Gly Phe Glu Gly Ile Phe Leu Val Val Ala Asn Pro Val Asp Ile 115 120 125 Leu Thr His Ala Thr Trp Lys Ile Ser Gly Phe Pro Lys Asp Arg Val 130 135 140 Ile Gly Ser Gly Thr Ser Leu Asp Thr Gly Arg Leu Gln Lys Val Ile 145 150 155 160 Gly Lys Met Glu His Val Asp Pro Arg Ser Val Asn Ala Tyr Met Leu 165 170 175 Gly Glu His Gly Asp Thr Glu Phe Pro Val Trp Ser Tyr Asn Asn Val 180 185 190 Gly Gly Val Lys Val Ser Asp Trp Val Lys Ala His Gly Met Asp Glu 195 200 205 Ser Lys Leu Glu Glu Ile His Lys Glu Val Ala Asp Met Ala Tyr Asp 210 215 220 Ile Ile Asn Lys Lys Gly Ala Thr Phe Tyr Gly Ile Gly Thr Ala Ser 225 230 235 240 Ala Met Ile Ala Lys Ala Ile Leu Asn Asp Glu His Arg Val Leu Pro 245 250 255 Leu Ser Val Ala Met Asp Gly Gln Tyr Gly Leu His Asp Leu His Ile 260 265 270 Gly Thr Pro Ala Val Val Gly Arg Asn Gly Leu Glu Gln Ile Ile Glu 275 280 285 Met Pro Leu Thr Ala Asp Glu Gln Ala Lys Met Glu Ala Ser Ala Lys 290 295 300 Gln Leu Lys Glu Val Met Asp Lys Ala Phe Glu Glu Thr Gly Val Lys 305 310 315 320 Val Arg Gln <210> SEQ ID NO 31 <211> LENGTH: 927 <212> TYPE: DNA <213> ORGANISM: Lactobacillus acidophilus <400> SEQUENCE: 31 atgagtagaa aagtgtttct tgtaggtgat ggtgctgttg gttcaacttt tgcaaatgac 60 ttattgcaaa atacaactgt tgatgaatta gcgatttttg atgttgctaa agatcgtcca 120 gttggtgatt caatggattt ggaagatatt actccattta caggtcaaac taatattcat 180 ccagcagaat atagtgatgc taaagatgca gatgtgtgtg taattactgc tggtgttcct 240 cgtaaacctg gtgaaactag acttgactta gttaataaga atgtaaagat tttaaagact 300 attgttgatc cggttgttga atccggtttt aagggtgtat ttgttgtttc agctaacccg 360 gttgatattt taaccacatt gactcaaaaa atatccggtt ttccaaaaga tcgtgtaatt 420 ggtactggta cttcacttga ttcaatgcgt cttcgcgttg aattggcaaa gaaacttaat 480 gttccagtag ctaaggttaa ctcaatggtt cttggtgaac acggtgatac tagttttgaa 540 aactttgacg aatcaactgt tgacaataag ccacttcgcg attactcaga aatcaatgat 600 aatgttttaa gtgaaattga gtcagacgtc cgtaaaaagg gtggaaagat catcactaac 660 aaaggagcta cattctatgg tgttgctatg atgcttactc aaattgttag tgctatttta 720 gataatcgtt caatttgttt gccattatca gccccaatta atggtgaata tggcattaag 780 catgatcttt acttaggtac tccaactata attaacggta atggtattga aaaagttatt 840 gaaactaaac tttcagatgt agaaaaagct aagatgatca attctgcaga taagatgcaa 900 gaagttttat caggtgttga aatgtaa 927 <210> SEQ ID NO 32 <211> LENGTH: 308 <212> TYPE: PRT <213> ORGANISM: Lactobacillus acidophilus <400> SEQUENCE: 32 Met Ser Arg Lys Val Phe Leu Val Gly Asp Gly Ala Val Gly Ser Thr 1 5 10 15 Phe Ala Asn Asp Leu Leu Gln Asn Thr Thr Val Asp Glu Leu Ala Ile 20 25 30

Phe Asp Val Ala Lys Asp Arg Pro Val Gly Asp Ser Met Asp Leu Glu 35 40 45 Asp Ile Thr Pro Phe Thr Gly Gln Thr Asn Ile His Pro Ala Glu Tyr 50 55 60 Ser Asp Ala Lys Asp Ala Asp Val Cys Val Ile Thr Ala Gly Val Pro 65 70 75 80 Arg Lys Pro Gly Glu Thr Arg Leu Asp Leu Val Asn Lys Asn Val Lys 85 90 95 Ile Leu Lys Thr Ile Val Asp Pro Val Val Glu Ser Gly Phe Lys Gly 100 105 110 Val Phe Val Val Ser Ala Asn Pro Val Asp Ile Leu Thr Thr Leu Thr 115 120 125 Gln Lys Ile Ser Gly Phe Pro Lys Asp Arg Val Ile Gly Thr Gly Thr 130 135 140 Ser Leu Asp Ser Met Arg Leu Arg Val Glu Leu Ala Lys Lys Leu Asn 145 150 155 160 Val Pro Val Ala Lys Val Asn Ser Met Val Leu Gly Glu His Gly Asp 165 170 175 Thr Ser Phe Glu Asn Phe Asp Glu Ser Thr Val Asp Asn Lys Pro Leu 180 185 190 Arg Asp Tyr Ser Glu Ile Asn Asp Asn Val Leu Ser Glu Ile Glu Ser 195 200 205 Asp Val Arg Lys Lys Gly Gly Lys Ile Ile Thr Asn Lys Gly Ala Thr 210 215 220 Phe Tyr Gly Val Ala Met Met Leu Thr Gln Ile Val Ser Ala Ile Leu 225 230 235 240 Asp Asn Arg Ser Ile Cys Leu Pro Leu Ser Ala Pro Ile Asn Gly Glu 245 250 255 Tyr Gly Ile Lys His Asp Leu Tyr Leu Gly Thr Pro Thr Ile Ile Asn 260 265 270 Gly Asn Gly Ile Glu Lys Val Ile Glu Thr Lys Leu Ser Asp Val Glu 275 280 285 Lys Ala Lys Met Ile Asn Ser Ala Asp Lys Met Gln Glu Val Leu Ser 290 295 300 Gly Val Glu Met 305 <210> SEQ ID NO 33 <211> LENGTH: 1050 <212> TYPE: DNA <213> ORGANISM: Lactobacillus acidophilus <400> SEQUENCE: 33 atggtcatac taataaattt tacggaggtt aaatttatga caaagatttt tgcttacgct 60 attcgtaaag acgaagaacc attcttaaac gaatggaagg aagctcacaa agatatcgat 120 gttgattaca ctgataaact tttgactcct gaaactgcaa agcttgctga aggtgcagac 180 ggtgttgttg tttaccaaca attagactac actcctgaaa cccttcaagc attggcagat 240 gctggcgtaa ctaagatgtc attacgtaac gttggtgtcg ataacatcga catggacaag 300 gccaaagaat taggctttga aatcactaat gttcctgttt actcaccaga cgctattgct 360 gaacatgctg ctattcaagc tgcacgtgta ttacgtcaag acaagcgcat ggacgaaaag 420 atggctaaac gtgatttacg ttgggcacca actatcggcc gtgaagttcg tgaccaagtt 480 gtcggtgttg ttggtactgg tcacattggt caagtattta tgaagattat ggaaggcttt 540 ggcgcaaaag ttattgctta cgatatcttc aagaaccctg aacttgaaaa gaagggttac 600 tacgttgatt cacttgatga cttgtacaag caagctgatg taatttcact tcacgtacca 660 gacgttccag ctaacgtaca catgattaac gatgaatcaa tcgccaaaat gaaggatggc 720 gttgtaatcg taaactgctc acgtggtcca cttgttgaca ctgatgcagt aattcgtggt 780 ttagactcag gcaagatctt cggcttcgtt atggatactt acgaaggcga agttggtgta 840 tttaacaagg actgggaagg taaagaattc ccagacgaac gcttggcaga cttaattgat 900 cgtccaaacg tattggtaac cccacacact gccttctaca ctactcacgc tgtacgtaac 960 atggttgtta aggcatttga caacaacttg gaattaatca agggcgaaaa accagattct 1020 ccagttgctt tggacaagaa caagttctaa 1050 <210> SEQ ID NO 34 <211> LENGTH: 349 <212> TYPE: PRT <213> ORGANISM: Lactobacillus acidophilus <400> SEQUENCE: 34 Met Val Ile Leu Ile Asn Phe Thr Glu Val Lys Phe Met Thr Lys Ile 1 5 10 15 Phe Ala Tyr Ala Ile Arg Lys Asp Glu Glu Pro Phe Leu Asn Glu Trp 20 25 30 Lys Glu Ala His Lys Asp Ile Asp Val Asp Tyr Thr Asp Lys Leu Leu 35 40 45 Thr Pro Glu Thr Ala Lys Leu Ala Glu Gly Ala Asp Gly Val Val Val 50 55 60 Tyr Gln Gln Leu Asp Tyr Thr Pro Glu Thr Leu Gln Ala Leu Ala Asp 65 70 75 80 Ala Gly Val Thr Lys Met Ser Leu Arg Asn Val Gly Val Asp Asn Ile 85 90 95 Asp Met Asp Lys Ala Lys Glu Leu Gly Phe Glu Ile Thr Asn Val Pro 100 105 110 Val Tyr Ser Pro Asp Ala Ile Ala Glu His Ala Ala Ile Gln Ala Ala 115 120 125 Arg Val Leu Arg Gln Asp Lys Arg Met Asp Glu Lys Met Ala Lys Arg 130 135 140 Asp Leu Arg Trp Ala Pro Thr Ile Gly Arg Glu Val Arg Asp Gln Val 145 150 155 160 Val Gly Val Val Gly Thr Gly His Ile Gly Gln Val Phe Met Lys Ile 165 170 175 Met Glu Gly Phe Gly Ala Lys Val Ile Ala Tyr Asp Ile Phe Lys Asn 180 185 190 Pro Glu Leu Glu Lys Lys Gly Tyr Tyr Val Asp Ser Leu Asp Asp Leu 195 200 205 Tyr Lys Gln Ala Asp Val Ile Ser Leu His Val Pro Asp Val Pro Ala 210 215 220 Asn Val His Met Ile Asn Asp Glu Ser Ile Ala Lys Met Lys Asp Gly 225 230 235 240 Val Val Ile Val Asn Cys Ser Arg Gly Pro Leu Val Asp Thr Asp Ala 245 250 255 Val Ile Arg Gly Leu Asp Ser Gly Lys Ile Phe Gly Phe Val Met Asp 260 265 270 Thr Tyr Glu Gly Glu Val Gly Val Phe Asn Lys Asp Trp Glu Gly Lys 275 280 285 Glu Phe Pro Asp Glu Arg Leu Ala Asp Leu Ile Asp Arg Pro Asn Val 290 295 300 Leu Val Thr Pro His Thr Ala Phe Tyr Thr Thr His Ala Val Arg Asn 305 310 315 320 Met Val Val Lys Ala Phe Asp Asn Asn Leu Glu Leu Ile Lys Gly Glu 325 330 335 Lys Pro Asp Ser Pro Val Ala Leu Asp Lys Asn Lys Phe 340 345 <210> SEQ ID NO 35 <211> LENGTH: 5323 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: vector <400> SEQUENCE: 35 gaattcagat ctctcgagcc cgggatcgat ggtacctcgc gaaagcttgg atgttgtaca 60 ggataatgtc cagaaggtcg atagaaagcg tgagaaacag cgtacagacg atttagagat 120 gtagaggtac ttttatgccg agaaaacttt ttgcgtgtga cagtccttaa aatatactta 180 gagcgtaagc gaaagtagta gcgacagcta ttaactttcg gttgcaaagc tctaggattt 240 ttaatggacg cagcgcatca cacgcaaaaa ggaaattgga ataaatgcga aatttgagat 300 gttaattaaa gacctttttg aggtcttttt ttcttagatt tttggggtta tttaggggag 360 aaaacatagg ggggtactac gacctccccc ctaggtgtcc attgtccatt gtccaaacaa 420 ataaataaat attgggtttt taatgttaaa aggttgtttt ttatgttaaa gtgaaaaaaa 480 cagatgttgg gaggtacagt gatagttgta gatagaaaag aagagaaaaa agttgctgtt 540 actttaagac ttacaacaga agaaaatgag atattaaata gaatcaaaga aaaatataat 600 attagcaaat cagatgcaac cggtattcta ataaaaaaat atgcaaagga ggaatacggt 660 gcattttaaa caaaaaaaga tagacagcac tggcatgctg cctatctatg actaaatttt 720 gttaagtgta ttagcaccgt tattatatca tgagcgaaaa tgtaataaaa gaaactgaaa 780 acaagaaaaa ttcaagagga cgtaattgga catttgtttt atatccagaa tcagcaaaag 840 ccgagtggtt agagtattta aaagagttac acattcaatt tgtagtgtct ccattacatg 900 atagggatac tgatacagaa ggtaggatga aaaaagagca ttatcatatt ctagtgatgt 960 atgagggtaa taaatcttat gaacagataa aaataattaa cagaagaatt gaatgcgact 1020 attccgcaga ttgcaggaag tgtgaaaggt cttgtgagat atatgcttca catggacgat 1080 cctaataaat ttaaatatca aaaagaagat atgatagttt atggcggtgt agatgttgat 1140 gaattattaa agaaaacaac aacagataga tataaattaa ttaaagaaat gattgagttt 1200 attgatgaac aaggaatcgt agaatttaag agtttaatgg attatgcaat gaagtttaaa 1260 tttgatgatt ggttcccgct tttatgtgat aactcggcgt atgttattca agaatatata 1320 aaatcaaatc ggtataaatc tgaccgatag attttgaatt taggtgtcac aagacactct 1380 tttttcgcac cagcgaaaac tggtttaagc cgactgcgca aaagacataa tcgattcaca 1440 aaaaataggc acacgaaaaa caagttaagg gatgcagttt atgcatccct taacttactt 1500 attaaataat ttatagctat tgaaaagaga taagaattgt tcaaagctaa tattgtttaa 1560 atcgtcaatt cctgcatgtt ttaaggaatt gttaaattga ttttttgtaa atattttctt 1620 gtattctttg ttaacccatt tcataacgaa ataattatac ttttgtttat ctttgtgtga 1680 tattcttgat ttttttctac ttaatctgat aagtgagcta ttcactttag gtttaggatg 1740 aaaatattct cttggaacca tacttaatat agaaatatca acttctgcca ttaaaagtaa 1800 tgccaatgag cgttttgtat ttaataatct tttagcaaac ccgtattcca cgattaaata 1860 aatctcatta gctatactat caaaaacaat tttgcgtatt atatccgtac ttatgttata 1920 aggtatatta ccatatattt tataggattg gtttttagga aatttaaact gcaatatatc 1980 cttgtttaaa acttggaaat tatcgtgatc aacaagttta ttttctgtag ttttgcataa 2040

tttatggtct atttcaatgg cagttacgaa attacacctc tttactaatt caagggtaaa 2100 atggcctttt cctgagccga tttcaaagat attatcatgt tcatttaatc ttatatttgt 2160 cattatttta tctatattat gttttgaagt aataaagttt tgactgtgtt ttatattttt 2220 ctcgttcatt ataaccctct ttaatttggt tatatgaatt ttgcttatta acgattcatt 2280 ataaccactt attttttgtt tggttgataa tgaactgtgc tgattacaaa aatactaaaa 2340 atgcccatat tttttcctcc ttataaaatt agtataatta tagcacgagc tctgataaat 2400 atgaacatga tgagtgatcg ttaaatttat actgcaatcg gatgcgatta ttgaataaaa 2460 gatatgagag atttatctaa tttctttttt cttgtaaaaa aagaaagttc ttaaaggttt 2520 tatagttttg gtcgtagagc acacggttta acgacttaat tacgaagtaa ataagtctag 2580 tgtgttagac tttatgaaat ctatatacgt ttatatatat ttattatccg gatctgcatc 2640 gcaggatgct gctggctacc ctgtggaaca cctacatctg tattaacgaa gcgctggcat 2700 tgaccctgag tgatttttct ctggtcccgc cgcatccata ccgccagttg tttaccctca 2760 caacgttcca gtaaccgggc atgttcatca tcagtaaccc gtatcgtgag catcctctct 2820 cgtttcatcg gtatcattac ccccatgaac agaaattccc ccttacacgg aggcatcaag 2880 tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt tatcagaagc cagacattaa 2940 cgcttctgga gaaactcaac gagctggacg cggatgaaca ggcagacatc tgtgaatcgc 3000 ttcacgacca cgctgatgag ctttaccgca gctgcctcgc gcgtttcggt gatgacggtg 3060 aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg 3120 ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca 3180 tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg catcagagca 3240 gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg taaggagaaa 3300 ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 3360 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 3420 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 3480 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 3540 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 3600 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 3660 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc 3720 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 3780 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 3840 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 3900 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc 3960 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 4020 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 4080 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 4140 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 4200 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 4260 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 4320 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 4380 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 4440 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 4500 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 4560 tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 4620 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 4680 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 4740 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 4800 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 4860 ttgcccggcg tcaacacggg ataataccgc gccacatagc agaactttaa aagtgctcat 4920 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 4980 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 5040 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 5100 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 5160 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 5220 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 5280 aacctataaa aataggcgta tcacgaggcc ctttcgtctt caa 5323 <210> SEQ ID NO 36 <211> LENGTH: 5581 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: vector <400> SEQUENCE: 36 gaattcacta gtcttaagta agtcgtattg gcaccactac tcacaccgtg accgacgcgc 60 ccgccagtca agtgttcaaa agttagcgtt tattaagtgc gataagtata ccacaaaggg 120 cttattgacg cccgccaaag ggttttgcgg acattgttaa taattgtatt aaaagcatgc 180 tcaatctaac acttattttg cacaaacatg gtatacttta accgtaaaaa ctaaattttc 240 actacgagag gatgacttat tttgtcaagc ctcgagcccg ggatcgatgg tacctcgcga 300 aagcttggat gttgtacagg ataatgtcca gaaggtcgat agaaagcgtg agaaacagcg 360 tacagacgat ttagagatgt agaggtactt ttatgccgag aaaacttttt gcgtgtgaca 420 gtccttaaaa tatacttaga gcgtaagcga aagtagtagc gacagctatt aactttcggt 480 tgcaaagctc taggattttt aatggacgca gcgcatcaca cgcaaaaagg aaattggaat 540 aaatgcgaaa tttgagatgt taattaaaga cctttttgag gtcttttttt cttagatttt 600 tggggttatt taggggagaa aacatagggg ggtactacga cctcccccct aggtgtccat 660 tgtccattgt ccaaacaaat aaataaatat tgggttttta atgttaaaag gttgtttttt 720 atgttaaagt gaaaaaaaca gatgttggga ggtacagtga tagttgtaga tagaaaagaa 780 gagaaaaaag ttgctgttac tttaagactt acaacagaag aaaatgagat attaaataga 840 atcaaagaaa aatataatat tagcaaatca gatgcaaccg gtattctaat aaaaaaatat 900 gcaaaggagg aatacggtgc attttaaaca aaaaaagata gacagcactg gcatgctgcc 960 tatctatgac taaattttgt taagtgtatt agcaccgtta ttatatcatg agcgaaaatg 1020 taataaaaga aactgaaaac aagaaaaatt caagaggacg taattggaca tttgttttat 1080 atccagaatc agcaaaagcc gagtggttag agtatttaaa agagttacac attcaatttg 1140 tagtgtctcc attacatgat agggatactg atacagaagg taggatgaaa aaagagcatt 1200 atcatattct agtgatgtat gagggtaata aatcttatga acagataaaa ataattaaca 1260 gaagaattga atgcgactat tccgcagatt gcaggaagtg tgaaaggtct tgtgagatat 1320 atgcttcaca tggacgatcc taataaattt aaatatcaaa aagaagatat gatagtttat 1380 ggcggtgtag atgttgatga attattaaag aaaacaacaa cagatagata taaattaatt 1440 aaagaaatga ttgagtttat tgatgaacaa ggaatcgtag aatttaagag tttaatggat 1500 tatgcaatga agtttaaatt tgatgattgg ttcccgcttt tatgtgataa ctcggcgtat 1560 gttattcaag aatatataaa atcaaatcgg tataaatctg accgatagat tttgaattta 1620 ggtgtcacaa gacactcttt tttcgcacca gcgaaaactg gtttaagccg actgcgcaaa 1680 agacataatc gattcacaaa aaataggcac acgaaaaaca agttaaggga tgcagtttat 1740 gcatccctta acttacttat taaataattt atagctattg aaaagagata agaattgttc 1800 aaagctaata ttgtttaaat cgtcaattcc tgcatgtttt aaggaattgt taaattgatt 1860 ttttgtaaat attttcttgt attctttgtt aacccatttc ataacgaaat aattatactt 1920 ttgtttatct ttgtgtgata ttcttgattt ttttctactt aatctgataa gtgagctatt 1980 cactttaggt ttaggatgaa aatattctct tggaaccata cttaatatag aaatatcaac 2040 ttctgccatt aaaagtaatg ccaatgagcg ttttgtattt aataatcttt tagcaaaccc 2100 gtattccacg attaaataaa tctcattagc tatactatca aaaacaattt tgcgtattat 2160 atccgtactt atgttataag gtatattacc atatatttta taggattggt ttttaggaaa 2220 tttaaactgc aatatatcct tgtttaaaac ttggaaatta tcgtgatcaa caagtttatt 2280 ttctgtagtt ttgcataatt tatggtctat ttcaatggca gttacgaaat tacacctctt 2340 tactaattca agggtaaaat ggccttttcc tgagccgatt tcaaagatat tatcatgttc 2400 atttaatctt atatttgtca ttattttatc tatattatgt tttgaagtaa taaagttttg 2460 actgtgtttt atatttttct cgttcattat aaccctcttt aatttggtta tatgaatttt 2520 gcttattaac gattcattat aaccacttat tttttgtttg gttgataatg aactgtgctg 2580 attacaaaaa tactaaaaat gcccatattt tttcctcctt ataaaattag tataattata 2640 gcacgagctc tgataaatat gaacatgatg agtgatcgtt aaatttatac tgcaatcgga 2700 tgcgattatt gaataaaaga tatgagagat ttatctaatt tcttttttct tgtaaaaaaa 2760 gaaagttctt aaaggtttta tagttttggt cgtagagcac acggtttaac gacttaatta 2820 cgaagtaaat aagtctagtg tgttagactt tatgaaatct atatacgttt atatatattt 2880 attatccgga tctgcatcgc aggatgctgc tggctaccct gtggaacacc tacatctgta 2940 ttaacgaagc gctggcattg accctgagtg atttttctct ggtcccgccg catccatacc 3000 gccagttgtt taccctcaca acgttccagt aaccgggcat gttcatcatc agtaacccgt 3060 atcgtgagca tcctctctcg tttcatcggt atcattaccc ccatgaacag aaattccccc 3120 ttacacggag gcatcaagtg accaaacagg aaaaaaccgc ccttaacatg gcccgcttta 3180 tcagaagcca gacattaacg cttctggaga aactcaacga gctggacgcg gatgaacagg 3240 cagacatctg tgaatcgctt cacgaccacg ctgatgagct ttaccgcagc tgcctcgcgc 3300 gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt 3360 gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 3420 ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat actggcttaa 3480 ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca 3540 cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc 3600 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg 3660 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa 3720 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga 3780 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag 3840 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct 3900 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc aatgctcacg 3960

ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc 4020 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt 4080 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta 4140 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac 4200 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc 4260 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat 4320 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc 4380 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt 4440 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta 4500 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct 4560 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg 4620 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga 4680 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt 4740 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt 4800 taatagtttg cgcaacgttg ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt 4860 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat 4920 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc 4980 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc 5040 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat 5100 gcggcgaccg agttgctctt gcccggcgtc aacacgggat aataccgcgc cacatagcag 5160 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt 5220 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc 5280 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa 5340 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg 5400 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa 5460 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac 5520 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttca 5580 a 5581 <210> SEQ ID NO 37 <211> LENGTH: 7443 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: vector <400> SEQUENCE: 37 gaattcacta gtcttaagta agtcgtattg gcaccactac tcacaccgtg accgacgcgc 60 ccgccagtca agtgttcaaa agttagcgtt tattaagtgc gataagtata ccacaaaggg 120 cttattgacg cccgccaaag ggttttgcgg acattgttaa taattgtatt aaaagcatgc 180 tcaatctaac acttattttg cacaaacatg gtatacttta accgtaaaaa ctaaattttc 240 actacgagag gatgacttat tttgtcaagc ctcgaggcta gcatatatag gaggaatttt 300 tgtaatgaaa aaagtcgcac ttgttaccgg cgccggccag gggattggta aagctatcgc 360 ccttcgtctg gtgaaggatg gatttgccgt ggccattgcc gattataacg acgccaccgc 420 caaagcggtc gcctccgaaa tcaaccaggc cggcggccgc gccatggcgg tgaaagtgga 480 tgtttctgac cgcgaccagg tatttgccgc cgtcgaacag gcgcgcaaaa cgctgggcgg 540 cttcgacgtc atcgtcaaca acgccggcgt ggcgccatcc acgccgatcg agtccattac 600 cccggagatt gtcgacaaag tctacaacat caacgtcaaa ggggtgatct ggggcatcca 660 ggcggcggtc gaggccttta agaaagaggg tcacggcggg aaaatcatca acgcctgttc 720 ccaggccggc cacgtcggca acccggagct ggcggtatat agctcgagta aattcgccgt 780 acgcggctta acccagaccg ccgctcgcga cctcgcgccg ctgggcatca cggtcaacgg 840 ctactgcccg gggattgtca aaacgccaat gtgggccgaa attgaccgcc aggtgtccga 900 agccgccggt aaaccgctgg gctacggtac cgccgagttc gccaaacgca tcaccctcgg 960 ccgcctgtcc gagccggaag atgtcgccgc ctgcgtctcc tatcttgcca gcccggattc 1020 tgattatatg accggtcagt cattgctgat cgacggcggg atggtgttta actaagatat 1080 catatatagg aggaattttt gtaatgaaag ctctggttta tcacggtgac cacaagatct 1140 cgcttgaaga caagcccaag cccacccttc aaaagcccac ggatgtagta gtacgggttt 1200 tgaagaccac gatctgcggc acggatctcg gcatctacaa aggcaagaat ccagaggtcg 1260 ccgacgggcg catcctgggc catgaagggg taggcgtcat cgaggaagtg ggcgagagtg 1320 tcacgcagtt caagaaaggc gacaaggtcc tgatttcctg cgtcacttct tgcggctcgt 1380 gcgactactg caagaagcag ctttactccc attgccgcga cggcgggtgg atcctgggtt 1440 acatgatcga tggcgtgcag gccgaatacg tccgcatccc gcatgccgac aacagcctct 1500 acaagatccc ccagacaatt gacgacgaaa tcgccgtcct gctgagcgac atcctgccca 1560 ccggccacga aatcggcgtc cagtatggga atgtccagcc gggcgatgcg gtggctattg 1620 tcggcgcggg ccccgtcggc atgtccgtac tgttgaccgc ccagttctac tccccctcga 1680 ccatcatcgt gatcgacatg gacgagaatc gcctccagct cgccaaggag ctcggggcaa 1740 cgcacaccat caactccggc acggagaacg ttgtcgaagc cgtgcatagg attgcggcag 1800 agggagtcga tgttgcgatc gaggcggtgg gcataccggc gacttgggac atctgccagg 1860 agatcgtcaa gcccggcgcg cacatcgcca acgtcggcgt gcatggcgtc aaggttgact 1920 tcgagattca gaagctctgg atcaagaacc tgacgatcac cacgggactg gtgaacacga 1980 acacgacgcc catgctgatg aaggtcgcct cgaccgacaa gcttccgttg aagaagatga 2040 ttacccatcg cttcgagctg gccgagatcg agcacgccta tcaggtattc ctcaatggcg 2100 ccaaggagaa ggcgatgaag atcatcctct cgaacgcagg cgctgcctga ggtacctcgc 2160 gaaagcttgg atgttgtaca ggataatgtc cagaaggtcg atagaaagcg tgagaaacag 2220 cgtacagacg atttagagat gtagaggtac ttttatgccg agaaaacttt ttgcgtgtga 2280 cagtccttaa aatatactta gagcgtaagc gaaagtagta gcgacagcta ttaactttcg 2340 gttgcaaagc tctaggattt ttaatggacg cagcgcatca cacgcaaaaa ggaaattgga 2400 ataaatgcga aatttgagat gttaattaaa gacctttttg aggtcttttt ttcttagatt 2460 tttggggtta tttaggggag aaaacatagg ggggtactac gacctccccc ctaggtgtcc 2520 attgtccatt gtccaaacaa ataaataaat attgggtttt taatgttaaa aggttgtttt 2580 ttatgttaaa gtgaaaaaaa cagatgttgg gaggtacagt gatagttgta gatagaaaag 2640 aagagaaaaa agttgctgtt actttaagac ttacaacaga agaaaatgag atattaaata 2700 gaatcaaaga aaaatataat attagcaaat cagatgcaac cggtattcta ataaaaaaat 2760 atgcaaagga ggaatacggt gcattttaaa caaaaaaaga tagacagcac tggcatgctg 2820 cctatctatg actaaatttt gttaagtgta ttagcaccgt tattatatca tgagcgaaaa 2880 tgtaataaaa gaaactgaaa acaagaaaaa ttcaagagga cgtaattgga catttgtttt 2940 atatccagaa tcagcaaaag ccgagtggtt agagtattta aaagagttac acattcaatt 3000 tgtagtgtct ccattacatg atagggatac tgatacagaa ggtaggatga aaaaagagca 3060 ttatcatatt ctagtgatgt atgagggtaa taaatcttat gaacagataa aaataattaa 3120 cagaagaatt gaatgcgact attccgcaga ttgcaggaag tgtgaaaggt cttgtgagat 3180 atatgcttca catggacgat cctaataaat ttaaatatca aaaagaagat atgatagttt 3240 atggcggtgt agatgttgat gaattattaa agaaaacaac aacagataga tataaattaa 3300 ttaaagaaat gattgagttt attgatgaac aaggaatcgt agaatttaag agtttaatgg 3360 attatgcaat gaagtttaaa tttgatgatt ggttcccgct tttatgtgat aactcggcgt 3420 atgttattca agaatatata aaatcaaatc ggtataaatc tgaccgatag attttgaatt 3480 taggtgtcac aagacactct tttttcgcac cagcgaaaac tggtttaagc cgactgcgca 3540 aaagacataa tcgattcaca aaaaataggc acacgaaaaa caagttaagg gatgcagttt 3600 atgcatccct taacttactt attaaataat ttatagctat tgaaaagaga taagaattgt 3660 tcaaagctaa tattgtttaa atcgtcaatt cctgcatgtt ttaaggaatt gttaaattga 3720 ttttttgtaa atattttctt gtattctttg ttaacccatt tcataacgaa ataattatac 3780 ttttgtttat ctttgtgtga tattcttgat ttttttctac ttaatctgat aagtgagcta 3840 ttcactttag gtttaggatg aaaatattct cttggaacca tacttaatat agaaatatca 3900 acttctgcca ttaaaagtaa tgccaatgag cgttttgtat ttaataatct tttagcaaac 3960 ccgtattcca cgattaaata aatctcatta gctatactat caaaaacaat tttgcgtatt 4020 atatccgtac ttatgttata aggtatatta ccatatattt tataggattg gtttttagga 4080 aatttaaact gcaatatatc cttgtttaaa acttggaaat tatcgtgatc aacaagttta 4140 ttttctgtag ttttgcataa tttatggtct atttcaatgg cagttacgaa attacacctc 4200 tttactaatt caagggtaaa atggcctttt cctgagccga tttcaaagat attatcatgt 4260 tcatttaatc ttatatttgt cattatttta tctatattat gttttgaagt aataaagttt 4320 tgactgtgtt ttatattttt ctcgttcatt ataaccctct ttaatttggt tatatgaatt 4380 ttgcttatta acgattcatt ataaccactt attttttgtt tggttgataa tgaactgtgc 4440 tgattacaaa aatactaaaa atgcccatat tttttcctcc ttataaaatt agtataatta 4500 tagcacgagc tctgataaat atgaacatga tgagtgatcg ttaaatttat actgcaatcg 4560 gatgcgatta ttgaataaaa gatatgagag atttatctaa tttctttttt cttgtaaaaa 4620 aagaaagttc ttaaaggttt tatagttttg gtcgtagagc acacggttta acgacttaat 4680 tacgaagtaa ataagtctag tgtgttagac tttatgaaat ctatatacgt ttatatatat 4740 ttattatccg gatctgcatc gcaggatgct gctggctacc ctgtggaaca cctacatctg 4800 tattaacgaa gcgctggcat tgaccctgag tgatttttct ctggtcccgc cgcatccata 4860 ccgccagttg tttaccctca caacgttcca gtaaccgggc atgttcatca tcagtaaccc 4920 gtatcgtgag catcctctct cgtttcatcg gtatcattac ccccatgaac agaaattccc 4980 ccttacacgg aggcatcaag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt 5040 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca 5100 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc 5160 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc 5220 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg 5280 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt 5340 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 5400 cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac 5460 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 5520

cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 5580 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 5640 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 5700 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 5760 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcaatgctca 5820 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 5880 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 5940 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6000 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg 6060 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 6120 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 6180 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 6240 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 6300 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 6360 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 6420 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag 6480 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca 6540 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact 6600 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca 6660 gttaatagtt tgcgcaacgt tgttgccatt gctgcaggca tcgtggtgtc acgctcgtcg 6720 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc 6780 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg 6840 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca 6900 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt 6960 atgcggcgac cgagttgctc ttgcccggcg tcaacacggg ataataccgc gccacatagc 7020 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc 7080 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca 7140 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa 7200 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat 7260 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa 7320 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa 7380 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctt 7440 caa 7443 <210> SEQ ID NO 38 <211> LENGTH: 5311 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: vector <400> SEQUENCE: 38 gaattcacta gtcttaagta agtcgtattg gcaccactac tcacaccgtg accgacgcgc 60 ccgccagtca agtgttcaaa agttagcgtt tattaagtgc gataagtata ccacaaaggg 120 cttattgacg cccgccaaag ggttttgcgg acattgttaa taattgtatt aaaagcatgc 180 tcaatctaac acttattttg cacaaacatg gtatacttta accgtaaaaa ctaaattttc 240 actacgagag gatgacttat tttgtcaagc ctcgaggcta gcatatatag gaggaatttt 300 tgtaatgaaa aaagtcgcac ttgttaccgg cgccggccag gggattggta aagctatcgc 360 ccttcgtctg gtgaaggatg gatttgccgt ggccattgcc gattataacg acgccaccgc 420 caaagcggtc gcctccgaaa tcaaccaggc cggcggccgc gccatggcgg tgaaagtgga 480 tgtttctgac cgcgaccagg tatttgccgc cgtcgaacag gcgcgcaaaa cgctgggcgg 540 cttcgacgtc atcgtcaaca acgccggcgt ggcgccatcc acgccgatcg agtccattac 600 cccggagatt gtcgacaaag tctacaacat caacgtcaaa ggggtgatct ggggcatcca 660 ggcggcggtc gaggccttta agaaagaggg tcacggcggg aaaatcatca acgcctgttc 720 ccaggccggc cacgtcggca acccggagct ggcggtatat agctcgagta aattcgccgt 780 acgcggctta acccagaccg ccgctcgcga cctcgcgccg ctgggcatca cggtcaacgg 840 ctactgcccg gggattgtca aaacgccaat gtgggccgaa attgaccgcc aggtgtccga 900 agccgccggt aaaccgctgg gctacggtac cgccgagttc gccaaacgca tcaccctcgg 960 ccgcctgtcc gagccggaag atgtcgccgc ctgcgtctcc tatcttgcca gcccggattc 1020 tgattatatg accggtcagt cattgctgat cgacggcggg atggtgttta actaagatag 1080 cttggatgtt gtacaggata atgtccagaa ggtcgataga aagcgtgaga aacagcgtac 1140 agacgattta gagatgtaga ggtactttta tgccgagaaa actttttgcg tgtgacagtc 1200 cttaaaatat acttagagcg taagcgaaag tagtagcgac agctattaac tttcggttgc 1260 aaagctctag gatttttaat ggacgcagcg catcacacgc aaaaaggaaa ttggaataaa 1320 tgcgaaattt gagatgttaa ttaaagacct ttttgaggtc tttttttctt agatttttgg 1380 ggttatttag gggagaaaac ataggggggt actacgacct cccccctagg tgtccattgt 1440 ccattgtcca aacaaataaa taaatattgg gtttttaatg ttaaaaggtt gttttttatg 1500 ttaaagtgaa aaaaacagat gttgggaggt acagtgatag ttgtagatag aaaagaagag 1560 aaaaaagttg ctgttacttt aagacttaca acagaagaaa atgagatatt aaatagaatc 1620 aaagaaaaat ataatattag caaatcagat gcaaccggta ttctaataaa aaaatatgca 1680 aaggaggaat acggtgcatt ttaaacaaaa aaagatagac agcactggca tgctgcctat 1740 ctatgactaa attttgttaa gtgtattagc accgttatta tatcatgagc gaaaatgtaa 1800 taaaagaaac tgaaaacaag aaaaattcaa gaggacgtaa ttggacattt gttttatatc 1860 cagaatcagc aaaagccgag tggttagagt atttaaaaga gttacacatt caatttgtag 1920 tgtctccatt acatgatagg gatactgata cagaaggtag gatgaaaaaa gagcattatc 1980 atattctagt gatgtatgag ggtaataaat cttatgaaca gataaaaata attaacagaa 2040 gaattgaatg cgactattcc gcagattgca ggaagtgtga aaggtcttgt gagatatatg 2100 cttcacatgg acgatcctaa taaatttaaa tatcaaaaag aagatatgat agtttatggc 2160 ggtgtagatg ttgatgaatt attaaagaaa acaacaacag atagatataa attaattaaa 2220 gaaatgattg agtttattga tgaacaagga atcgtagaat ttaagagttt aatggattat 2280 gcaatgaagt ttaaatttga tgattggttc ccgcttttat gtgataactc ggcgtatgtt 2340 attcaagaat atataaaatc aaatcggtat aaatctgacc gatagatttt gaatttaggt 2400 gtcacaagac actctttttt cgcaccagcg aaaactggtt taagccgact gcgcaaaaga 2460 cataatcgat tcacaaaaaa taggcacacg aaaaacaagt taagggatgc agtttatgca 2520 tcccttaact tacttattaa ataatttata gctattgaaa agagataaga attgttcaaa 2580 gctaatattg tttaaatcgt caattcctgc atgttttaag gaattgttaa attgattttt 2640 tgtaaatatt ttcttgtatt ctttgttaac ccatttcata acgaaataat tatacttttg 2700 tttatctttg tgtgatattc ttgatttttt tctacttaat ctgataagtg agctattcac 2760 tttaggttta ggatgaaaat attctcttgg aaccatactt aatatagaaa tatcaacttc 2820 tgccattaaa agtaatgcca atgagcgttt tgtatttaat aatcttttag caaacccgta 2880 ttccacgatt aaataaatct cattagctat actatcaaaa acaattttgc gtattatatc 2940 cgtacttatg ttataaggta tattaccata tattttatag gattggtttt taggaaattt 3000 aaactgcaat atatccttgt ttaaaacttg gaaattatcg tgatcaacaa gtttattttc 3060 tgtagttttg cataatttat ggtctatttc aatggcagtt acgaaattac acctctttac 3120 taattcaagg gtaaaatggc cttttcctga gccgatttca aagatattat catgttcatt 3180 taatcttata tttgtcatta ttttatctat attatgtttt gaagtaataa agttttgact 3240 gtgttttata tttttctcgt tcattataac cctctttaat ttggttatat gaattttgct 3300 tattaacgat tcattataac cacttatttt ttgtttggtt gataatgaac tgtgctgatt 3360 acaaaaatac taaaaatgcc catatttttt cctccttata aaattagtat aattatagca 3420 cgagctctga taaatatgaa catgatgagt gatcgttaaa tttatactgc aatcggatgc 3480 gattattgaa taaaagatat gagagattta tctaatttct tttttcttgt aaaaaaagaa 3540 agttcttaaa ggttttatag ttttggtcgt agagcacacg gtttaacgac ttaattacga 3600 agtaaataag tctagtgtgt tagactttat gaaatctata tacgtttata tatatttatt 3660 atccggatct gcatcgcagg atgctgctgg ctaccctgtg gaacacctac atctgtatta 3720 acgaagcgct ggcattgacc ctgagtgatt tttctctggt cccgccgcat ccataccgcc 3780 agttgtttac cctcacaacg ttccagtaac cgggcatgtt catcatcagt aacccgtatc 3840 gtgagcatcc tctctcgttt catcggtatc attaccccca tgaacagaaa ttccccctta 3900 cacggaggca tcaagtgacc aaacaggaaa aaaccgccct taacatggcc cgctttatca 3960 gaagccagac attaacgctt ctggagaaac tcaacgagct ggacgcggat gaacaggcag 4020 acatctgtga atcgcttcac gaccacgctg atgagcttta ccgcagctgc ctcgcgcgtt 4080 tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 4140 tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 4200 gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta 4260 tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag 4320 atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct 4380 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 4440 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 4500 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 4560 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 4620 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 4680 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg 4740 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 4800 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 4860 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 4920 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 4980 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 5040 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 5100 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 5160 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac 5220 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac 5280

ttggtctgac agttaccaat gcttaatcag t 5311 <210> SEQ ID NO 39 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 39 catgaattcg tgctaagagc cagattgtgg a 31 <210> SEQ ID NO 40 <211> LENGTH: 50 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 40 catgaagacc acgcgtaggc cttctagagc taaattttca catcgtgagc 50 <210> SEQ ID NO 41 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 41 atttagctct agaaggccta cgcgtggtct tcatgaactt gttcaaccg 49 <210> SEQ ID NO 42 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 42 catctcgagc caagctcagt cacgcattta a 31 <210> SEQ ID NO 43 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 43 aagcacaacg ggaagcgaac at 22 <210> SEQ ID NO 44 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 44 atacaactat gacgctggaa gcg 23 <210> SEQ ID NO 45 <211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 45 gtaggttttc ccgtccttga tag 23 <210> SEQ ID NO 46 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 46 tataagatct tgactctggt gaacttgtcg caacc 35 <210> SEQ ID NO 47 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 47 atatctcgag aataagtcat cctctcgtag tgaa 34 <210> SEQ ID NO 48 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 48 tatactcgag taatcatttc atacgattaa atgt 34 <210> SEQ ID NO 49 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 49 atatcccggg gtgagcgggt aaagtccttg cc 32 <210> SEQ ID NO 50 <211> LENGTH: 69 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 50 tatactcgag gctagcatat gatatcatat ataggaggaa tttttgtaat gaaagctctg 60 gtttatcac 69 <210> SEQ ID NO 51 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 51 atatatagga ggaatttttg ta 22 <210> SEQ ID NO 52 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 52 tataggtacc tcaggcagcg cctgcgttcg agag 34 <210> SEQ ID NO 53 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 53 cgggcacctg caaccgaggt c 21 <210> SEQ ID NO 54 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 54 ctgtttctca cgctttctat cg 22 <210> SEQ ID NO 55 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 55 gattttcttt atcaacttcg ac 22 <210> SEQ ID NO 56 <211> LENGTH: 55 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 56 tatagctagc atatatagga ggaatttttg taatgaaaaa agtcgcactt gttac 55 <210> SEQ ID NO 57 <211> LENGTH: 31 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 57 tatagatatc ttagttaaac accatcccgc c 31 <210> SEQ ID NO 58 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 58 ttggaaaacg ttcttcgggg c 21 <210> SEQ ID NO 59 <211> LENGTH: 49 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE:

<223> OTHER INFORMATION: primer <400> SEQUENCE: 59 atgttgtaca aaacaggagg gccaaaatca tgggcaatta cgattcaac 49 <210> SEQ ID NO 60 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 60 atcctgtaca ctaaccaccg atttggcaat gtag 34 <210> SEQ ID NO 61 <211> LENGTH: 46 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 61 tagaattcgt gaattccact agtcttaagt aagtcgtatt ggcacc 46 <210> SEQ ID NO 62 <211> LENGTH: 47 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 62 acttgtagga ctcgaggctt gacaaaataa gtcatcctct cgtagtg 47 <210> SEQ ID NO 63 <211> LENGTH: 1053 <212> TYPE: DNA <213> ORGANISM: Bacillus cereus <400> SEQUENCE: 63 atgaaagcac tactttggca taatcaacgt gatgtacgag tagaagaagt accagaacca 60 acagtaaaac caggaacagt gaaaatcaaa gttaaatggt gtggtatttg tgggacagac 120 ttgcatgaat atttagcagg gcctattttt attccaacag aagaacatcc attaacacat 180 gtgaaagcac ctgttatttt aggtcatgag tttagtggtg aggtaataga gattggtgaa 240 ggagttacat ctcataaagt gggagaccgc gttgttgtag agccaattta ttcttgtggt 300 aaatgtgaag cttgtaaaca tggacattac aatgtttgtg aacaacttgt tttccacggt 360 cttggcggag aaggcggcgg tttctctgaa tatacagtag taccagaaga tatggttcat 420 cacattccag atgaaatgac gtatgaacaa ggtgcgcttg tagaaccagc agcagtagca 480 gttcatgcag tacgtcaaag taaattaaaa gaaggggaag ctgtagcggt atttggttgc 540 ggtccaattg gacttcttgt tatccaagca gctaaagcag caggagcaac tcctgttatt 600 gcagttgaac tttctaaaga acgtcaagag ttagcgaaat tagcaggtgc ggattatgta 660 ttaaatccag caactcaaga tgtgttagct gaaattcgta acttaacaaa tggtttaggt 720 gtaaatgtta gctttgaagt aacaggtgtt gaagttgtac tacgccaagc gattgaaagt 780 acaagcttcg aaggacaaac tgtaattgtt agtgtatggg aaaaagacgc aacaattact 840 ccaaataact tagtattaaa agaaaaagaa gttattggta ttttaggata ccgtcacatc 900 ttcccagctg ttattaaatt gattagctcc ggtcaaattc aagcagagaa attaattacg 960 aaaaaaatta cagtggatca agttgttgaa gaaggatttg aagcacttgt aaaagataaa 1020 acacaagtga aaattcttgt ttcacctaaa taa 1053 <210> SEQ ID NO 64 <211> LENGTH: 350 <212> TYPE: PRT <213> ORGANISM: Bacillus cereus <400> SEQUENCE: 64 Met Lys Ala Leu Leu Trp His Asn Gln Arg Asp Val Arg Val Glu Glu 1 5 10 15 Val Pro Glu Pro Thr Val Lys Pro Gly Thr Val Lys Ile Lys Val Lys 20 25 30 Trp Cys Gly Ile Cys Gly Thr Asp Leu His Glu Tyr Leu Ala Gly Pro 35 40 45 Ile Phe Ile Pro Thr Glu Glu His Pro Leu Thr His Val Lys Ala Pro 50 55 60 Val Ile Leu Gly His Glu Phe Ser Gly Glu Val Ile Glu Ile Gly Glu 65 70 75 80 Gly Val Thr Ser His Lys Val Gly Asp Arg Val Val Val Glu Pro Ile 85 90 95 Tyr Ser Cys Gly Lys Cys Glu Ala Cys Lys His Gly His Tyr Asn Val 100 105 110 Cys Glu Gln Leu Val Phe His Gly Leu Gly Gly Glu Gly Gly Gly Phe 115 120 125 Ser Glu Tyr Thr Val Val Pro Glu Asp Met Val His His Ile Pro Asp 130 135 140 Glu Met Thr Tyr Glu Gln Gly Ala Leu Val Glu Pro Ala Ala Val Ala 145 150 155 160 Val His Ala Val Arg Gln Ser Lys Leu Lys Glu Gly Glu Ala Val Ala 165 170 175 Val Phe Gly Cys Gly Pro Ile Gly Leu Leu Val Ile Gln Ala Ala Lys 180 185 190 Ala Ala Gly Ala Thr Pro Val Ile Ala Val Glu Leu Ser Lys Glu Arg 195 200 205 Gln Glu Leu Ala Lys Leu Ala Gly Ala Asp Tyr Val Leu Asn Pro Ala 210 215 220 Thr Gln Asp Val Leu Ala Glu Ile Arg Asn Leu Thr Asn Gly Leu Gly 225 230 235 240 Val Asn Val Ser Phe Glu Val Thr Gly Val Glu Val Val Leu Arg Gln 245 250 255 Ala Ile Glu Ser Thr Ser Phe Glu Gly Gln Thr Val Ile Val Ser Val 260 265 270 Trp Glu Lys Asp Ala Thr Ile Thr Pro Asn Asn Leu Val Leu Lys Glu 275 280 285 Lys Glu Val Ile Gly Ile Leu Gly Tyr Arg His Ile Phe Pro Ala Val 290 295 300 Ile Lys Leu Ile Ser Ser Gly Gln Ile Gln Ala Glu Lys Leu Ile Thr 305 310 315 320 Lys Lys Ile Thr Val Asp Gln Val Val Glu Glu Gly Phe Glu Ala Leu 325 330 335 Val Lys Asp Lys Thr Gln Val Lys Ile Leu Val Ser Pro Lys 340 345 350 <210> SEQ ID NO 65 <211> LENGTH: 1113 <212> TYPE: DNA <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 65 ttgcctgaaa cgacaaccat cctatataga ggaggcgttt ttatgcgcgc agcacgtttt 60 tacgaccgcg gggatatccg cattgatgaa attaatgaac caatagtaaa agctggccaa 120 gttggcattg atgtggcttg gtgtggaatt tgtggaacag atctccatga atttttagat 180 ggcccaattt tttgtccgtc agcagaacat cctaatccaa ttactggaga agtaccacca 240 gtcactcttg gacatgaaat gtctggggtt gtaaatttta taggtgaagg agtaagcgga 300 cttaaagtag gtgaccatgt cgttgtcgaa ccttatatcg ttcccgaagg gactgataca 360 agtgaaactg gacattataa cctctcagaa ggctcaaact ttattggttt gggcggaaat 420 ggtggaggtt tggctgaaaa aatttctgtt gatgaacgtt gggttcacaa aattcctgat 480 aacttaccat tggatgaagc tgctctaatt gagccactat cagtcggcta tcacgctgtt 540 gaacgagcaa atttaagtga aaagagtacg gtattagttg ttggtgctgg accaattgga 600 ctattaactg ctgccgttgc aaaagcgcaa ggacatactg ttatcatcag tgaacctagt 660 ggacttcgtc gtaaaaaagc acaagaagca caagttgctg attatttctt caatccaatt 720 gaagatgaca ttcaagctaa agttcatgaa attaatgaaa aaggagtgga cgcagccttt 780 gaatgtacct ctgtccaacc gggatttgac gcttgtctag atgcgattcg tatgggtgga 840 acagttgtca ttgtcgcaat ttggggcaag cctgctagtg ttgatatggc aaaattagta 900 atcaaagaag ctaacctttt aggaacgatt gcttataata acactcatcc aaaaacaatt 960 gatttagtat caacaggtaa aataaaattg gaccaattca tcacagctaa aatcggtttg 1020 gatgatttga ttgataaagg attcgatacg ctgattcatc ataatgaaac agctgttaaa 1080 attttagttt caccaactgg taaaggtcta taa 1113 <210> SEQ ID NO 66 <211> LENGTH: 370 <212> TYPE: PRT <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 66 Met Pro Glu Thr Thr Thr Ile Leu Tyr Arg Gly Gly Val Phe Met Arg 1 5 10 15 Ala Ala Arg Phe Tyr Asp Arg Gly Asp Ile Arg Ile Asp Glu Ile Asn 20 25 30 Glu Pro Ile Val Lys Ala Gly Gln Val Gly Ile Asp Val Ala Trp Cys 35 40 45 Gly Ile Cys Gly Thr Asp Leu His Glu Phe Leu Asp Gly Pro Ile Phe 50 55 60 Cys Pro Ser Ala Glu His Pro Asn Pro Ile Thr Gly Glu Val Pro Pro 65 70 75 80 Val Thr Leu Gly His Glu Met Ser Gly Val Val Asn Phe Ile Gly Glu 85 90 95 Gly Val Ser Gly Leu Lys Val Gly Asp His Val Val Val Glu Pro Tyr 100 105 110 Ile Val Pro Glu Gly Thr Asp Thr Ser Glu Thr Gly His Tyr Asn Leu 115 120 125 Ser Glu Gly Ser Asn Phe Ile Gly Leu Gly Gly Asn Gly Gly Gly Leu 130 135 140 Ala Glu Lys Ile Ser Val Asp Glu Arg Trp Val His Lys Ile Pro Asp 145 150 155 160 Asn Leu Pro Leu Asp Glu Ala Ala Leu Ile Glu Pro Leu Ser Val Gly 165 170 175 Tyr His Ala Val Glu Arg Ala Asn Leu Ser Glu Lys Ser Thr Val Leu 180 185 190 Val Val Gly Ala Gly Pro Ile Gly Leu Leu Thr Ala Ala Val Ala Lys

195 200 205 Ala Gln Gly His Thr Val Ile Ile Ser Glu Pro Ser Gly Leu Arg Arg 210 215 220 Lys Lys Ala Gln Glu Ala Gln Val Ala Asp Tyr Phe Phe Asn Pro Ile 225 230 235 240 Glu Asp Asp Ile Gln Ala Lys Val His Glu Ile Asn Glu Lys Gly Val 245 250 255 Asp Ala Ala Phe Glu Cys Thr Ser Val Gln Pro Gly Phe Asp Ala Cys 260 265 270 Leu Asp Ala Ile Arg Met Gly Gly Thr Val Val Ile Val Ala Ile Trp 275 280 285 Gly Lys Pro Ala Ser Val Asp Met Ala Lys Leu Val Ile Lys Glu Ala 290 295 300 Asn Leu Leu Gly Thr Ile Ala Tyr Asn Asn Thr His Pro Lys Thr Ile 305 310 315 320 Asp Leu Val Ser Thr Gly Lys Ile Lys Leu Asp Gln Phe Ile Thr Ala 325 330 335 Lys Ile Gly Leu Asp Asp Leu Ile Asp Lys Gly Phe Asp Thr Leu Ile 340 345 350 His His Asn Glu Thr Ala Val Lys Ile Leu Val Ser Pro Thr Gly Lys 355 360 365 Gly Leu 370 <210> SEQ ID NO 67 <211> LENGTH: 3378 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: synthesized double coding region <400> SEQUENCE: 67 ggatccgttt aaacaggagg gccaaaatca tgggcaatta cgattcaaca ccgatagcta 60 aaagtgatag gattaaaaga ttggttgatc atttgtatgc taaaatgcct gaaattgagg 120 ccgctagagc agagctaatt actgaatcct ttaaggccac cgaaggtcaa cctgttgtta 180 tgagaaaggc tagagctttt gaacatatac taaagaattt gccaattatc ataagaccag 240 aagaactgat tgttggctca actacaattg cccctagagg ttgccaaacg tatccagaat 300 tctcatacga gtggttagag gctgaatttg aaactgtcga aacgcgttca gctgacccat 360 tttatatttc agaagaaacg aagaaacgtt tgctggctgc cgatgcttat tggaaaggta 420 aaacaacctc agagttggca acttcatata tggccccaga aactctaaga gccatgaagc 480 ataacttctt cacccctgga aactacttct acaatggtgt cggtcatgtc acagttcaat 540 atgaaacagt attagcaatc ggcttgaatg gagtaaaaga gaaggttagg aaagagatgg 600 agaattgtca ttttggtgat gccgattata gtacaaagat gtgtttcttg gagagcattt 660 taatatcgtg tgatgccgta atcacttatg ctaatagata tgccaagatg gccgaggaaa 720 tggctgaaaa agaaacagat gctgcaagga ggcaagaact attaacaatc gccagggttt 780 gcaaaaacgt tcctgaattc ccagccgaaa gcttccagga ggcctgccaa tccttttggt 840 tcatacaaca agtgcttcaa attgaatcca gtggtcattc aatttcccca ggtagatttg 900 atcaatatat gtatccttat tacgaaaagg atttaaagga aggtagctta actagggaat 960 atgctcagga actgatcgat tgtatctggg ttaagttaaa tgatctgaat aagtgcaggg 1020 atgctgcctc tgctgagggc tttgcaggat attccttatt tcaaaactta atcgttgggg 1080 gccaaacggt tcaaggaagg gacgccacca atgatttgag ttttatgtgt atcacggcat 1140 ctgaacacgt ctttttaccg atgccgtcgt tgtctataag agtttggcat ggtagttcca 1200 aagcactgct tatgagagca gctgaattga ctagaaccgg tataggctta cctgcttatt 1260 acaatgatga agtcatcata ccagctttgg tgcatagggg tgctactatg gatgaagcaa 1320 gaaattacaa cataatagga tgtgtcgaac cgcaggttcc tggtaaaact gatggctggc 1380 acgatgcagc attctttaac atgtgcagac ctttggaaat ggtgtttagt aatggttatg 1440 ataacggtga aattgcatct atacaaactg gtaacgtaga atcttttcag agttttgatg 1500 agtttatgga agcttacaga aaacaaatgc tatataacat agaacttatg gtaaatgccg 1560 acaacgcgat agattatgcc cacgcaaagt tggccccatt gccatttgag tcatgtttgg 1620 ttgatgactg tataaagaga ggaatgtccg ctcaggaagg cggcgcaatc tataatttca 1680 ctggtccaca gggctttggt attgcaaacg ttgctgatag cttgtatacg attaagaaat 1740 tggtgttcga ggagaagaga attacgatgg gtgaattaaa gaaagcgttg gaaatgaatt 1800 atggtaaggg tttggatgcc acaaccgctg gtgacatcgc aatgcaggtc gcgaagggac 1860 taaaagatgc cggacaggaa gtgggtcccg acgtgatcgc taatacaatc cgtcaagttc 1920 ttgaaatgga attaccagaa gatgtaagaa agagatatga agagatccat gaaatgatac 1980 ttgagttacc aaagtatggt aatgatatag atgaagttga tgaattagct agagaagcag 2040 cttactttta cacaagacca ttagaaactt ttaagaatcc aaggggtggc atgtatcaag 2100 ccggccttta tcccgtgtcc gctaatgtgc cactaggcgc tcaaacgggg gccacacccg 2160 atggacgttt ggcgcataca cccgtggcgg atggcgttgg tccgacatca ggcttcgata 2220 tatccggacc aacagcttct tgcaattctg tcgccaagtt ggatcatgct atagcctcta 2280 atggtacctt atttaatatg aagatgcacc caaccgcaat ggcaggtgaa aagggcttag 2340 aatccttcat atcgttgatc cgtggttatt tcgatcaaca aggtatgcac atgcaattta 2400 acgtagtaga cagggctaca ctgcttgatg cgcaggccca ccctgaaaag tattcaggct 2460 taattgtcag agtggcaggt tattctgccc tttttaccac attgtccaag tcattacaag 2520 atgatataat caaacgtacc gaacaagcag acaatagata ggaaggaaaa acgcgttatg 2580 aaagaatatc ttaatacttc aggtagaata tttgatatcc agaggtattc tattcacgat 2640 ggccctggtg tgcgtacaat tgtgtttcta aaaggttgtg cccttagatg cagatggtgc 2700 tgtaatcctg aaagccaaag cttcgaagtt gaaacaatga cgattaatgg aaaacctaaa 2760 gtcatgggta aagatgttac agtcgccgag gttatgaaga cggtagaaag agacatgcct 2820 tattaccttc aatcaggtgg tggtatcacc ttatcgggtg gcgaatgtac tttgcaacca 2880 gaattttccc ttggcctatt gagagctgca aaggatttgg gcatatccac ggcaatagag 2940 agcatggcgt acgcaaagta cgaagtaata gaaactcttc ttccgtattt ggatacgtat 3000 ttaatggaca tcaaacatat gaatcctgag aaacataaag aatacactgg tcatgataac 3060 ttgaggatgt tagaaaacgc cttaagagtc gcgcattctg gtcagaccga actgatcatc 3120 agagtacctg tcatcccagg attcaacgca actgagcagg aactactaga tattgcaaaa 3180 ttcgcagata cactgcctgg agttagacaa atacacatct tgccatatca taattttggt 3240 cagggtaaat acgaaggatt gaacagggac tatccgatgg gggacactga gaaaccctct 3300 aatgaacaga tgaaagcttt tcaagaaatg attcaaaaga acacttccct acattgccaa 3360 atcggtggtt aggtcgac 3378 <210> SEQ ID NO 68 <400> SEQUENCE: 68 000 <210> SEQ ID NO 69 <211> LENGTH: 2445 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 69 atgatcatgt ctgaaacttt aactaaaaca acgacaacta ttaaccactt cggtaaattg 60 acgccaatga tggatcgctt acgcgatagc atcattgatg caaaacctta tgtcgatcca 120 gaacgggcga ttctcacaac cgaaacttat cgacaacacc aagacgaaca agtcgatata 180 ttacgggcta aaatgcttga acacgttctt gataaaatga gtatcttcat tgaagatgat 240 actttaattg ttggtaacca agcacgccaa aatcgttggg caccagtatt ccctgagtat 300 tctatgaatt gggtcattga tgaattagat acatttgaga agcgtcctgg tgacgttttc 360 tatattacgg agaaatccaa ggaagaactt cgtgcgattg cgcctttctg gaaacataat 420 accttggaag accgcggcta cgctagtttt ccagaagcaa gtcgtatttt ttatgattta 480 ggtattattg gagccgatgg taatatcact tctggtgatg gtcacattgc ggtcgactat 540 aaaaacgttg ttaataaggg acttaaatgg tatgaagacc gcattaagac agcacttgct 600 aatcttgacc ttactgattt taaccagcaa aaacaatact atttctataa agcgggccta 660 attgtaattg atgccattca caattttgct aaacgttacg cccaattagc gtccaagcaa 720 gctcaaaaca cgacatccgc aactcgcaaa gcacaacttg aaaaaatcgc ccaaattcta 780 aacaaggttc cttacgaacc tgcaaattca ttttatgaag cgattcaagc tgtctggtta 840 gttcatctga ccttacaaat cgaatccaac ggtcattctg tctcatatgg tcgtctagat 900 cagtacctag ctccattcta tgagcacgat ttaaaaactg gtgctattga cgccaacggt 960 gcaaccgaat tactcacaaa cttatgtctt aagacgttaa cgattaataa agtacgctca 1020 tggcaacata ctgaattttc tgcagggagt cccctctacc aaaacattac gattggtggt 1080 caaacaccag atggtaaaga tgccgttaat ccgacgtcct atctgatttt acgagcaatt 1140 gcgcaagcac atttaccaca acccaactta acggtccgtt atcaccatgg cttaagcgat 1200 aagtttatgc gtgaatgtgt cgaagttatt aaacaaggct taggtatgcc tgcgtttaat 1260 aacgacgaaa ttattattcc gtcgtttatt cgtcgtggcg tcaagaaaga agacgcctat 1320 aattacagtg ccatcggttg tgtcgaaaca gcgatccctg gaaaatgggg ctatcgttgc 1380 accgggatga gcttcattaa cttcccacgc gttctcttac tcattatgaa tggtggcatt 1440 gatcctgaat ctggcaaacg gttattaccc gattatggta agttcactga tatgacttct 1500 tttgatcaac ttatgactgc ttgggacaaa gcgctccgtg aaatgacacg acaaagtgtg 1560 attatcgaaa atagttgtga tttggctttg gaacaaaatt atcctgatat tctctgctcc 1620 gttttaaccg acgattgtat cggtcgtggt aagaccatta aagaaggtgg cgcggtatac 1680 gactttatca gtggattaca agttggtatt gctaacctag cggactccct agctgcaatc 1740 aagaaacttg tctttgaaga aaagaagttg acaacaaccc aactttggca cgcacttacc 1800 actgattttg cggatgaaga tggtgaaaag attcggcaga tgctcattaa tgatgcccca 1860 aagtatggta acgatgatga ttatgttgat gatttgattg ttgaagctta taaaccatat 1920 attgatgaaa ttgccaagta caaaaacacg cgctacggtc gcggccctat tggtggcttg 1980 cgctacgcag gaacctcttc tatttcggcc aacgttggtc aagggcacag cactttggct 2040 acaccagatg gtcggcacgc tcggacacca ttagccgaag gttgctcacc agaacatgca 2100 atggatactg atggcccaac tgctgtgttc aaatcagttt ccaaattatc cactaaggac 2160 atcactggtg gcgtattact gaaccaaaag atgtcaccac aaattctacg gagtgatgaa 2220 agctgcatga aattggttgc actactacgg accttcttca atcgacttca tggttaccat 2280 gtccaataca acattgtttc acgggatacc ttgattgatg cacagaacca tcctgacaag 2340

caccgtgact tgattgttcg ggttgctgga tattccgcct tcttcgtggg cctatccaaa 2400 gaaacccaag atgatattat cgaacggacg gagcagtctc tataa 2445 <210> SEQ ID NO 70 <211> LENGTH: 814 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 70 Met Ile Met Ser Glu Thr Leu Thr Lys Thr Thr Thr Thr Ile Asn His 1 5 10 15 Phe Gly Lys Leu Thr Pro Met Met Asp Arg Leu Arg Asp Ser Ile Ile 20 25 30 Asp Ala Lys Pro Tyr Val Asp Pro Glu Arg Ala Ile Leu Thr Thr Glu 35 40 45 Thr Tyr Arg Gln His Gln Asp Glu Gln Val Asp Ile Leu Arg Ala Lys 50 55 60 Met Leu Glu His Val Leu Asp Lys Met Ser Ile Phe Ile Glu Asp Asp 65 70 75 80 Thr Leu Ile Val Gly Asn Gln Ala Arg Gln Asn Arg Trp Ala Pro Val 85 90 95 Phe Pro Glu Tyr Ser Met Asn Trp Val Ile Asp Glu Leu Asp Thr Phe 100 105 110 Glu Lys Arg Pro Gly Asp Val Phe Tyr Ile Thr Glu Lys Ser Lys Glu 115 120 125 Glu Leu Arg Ala Ile Ala Pro Phe Trp Lys His Asn Thr Leu Glu Asp 130 135 140 Arg Gly Tyr Ala Ser Phe Pro Glu Ala Ser Arg Ile Phe Tyr Asp Leu 145 150 155 160 Gly Ile Ile Gly Ala Asp Gly Asn Ile Thr Ser Gly Asp Gly His Ile 165 170 175 Ala Val Asp Tyr Lys Asn Val Val Asn Lys Gly Leu Lys Trp Tyr Glu 180 185 190 Asp Arg Ile Lys Thr Ala Leu Ala Asn Leu Asp Leu Thr Asp Phe Asn 195 200 205 Gln Gln Lys Gln Tyr Tyr Phe Tyr Lys Ala Gly Leu Ile Val Ile Asp 210 215 220 Ala Ile His Asn Phe Ala Lys Arg Tyr Ala Gln Leu Ala Ser Lys Gln 225 230 235 240 Ala Gln Asn Thr Thr Ser Ala Thr Arg Lys Ala Gln Leu Glu Lys Ile 245 250 255 Ala Gln Ile Leu Asn Lys Val Pro Tyr Glu Pro Ala Asn Ser Phe Tyr 260 265 270 Glu Ala Ile Gln Ala Val Trp Leu Val His Leu Thr Leu Gln Ile Glu 275 280 285 Ser Asn Gly His Ser Val Ser Tyr Gly Arg Leu Asp Gln Tyr Leu Ala 290 295 300 Pro Phe Tyr Glu His Asp Leu Lys Thr Gly Ala Ile Asp Ala Asn Gly 305 310 315 320 Ala Thr Glu Leu Leu Thr Asn Leu Cys Leu Lys Thr Leu Thr Ile Asn 325 330 335 Lys Val Arg Ser Trp Gln His Thr Glu Phe Ser Ala Gly Ser Pro Leu 340 345 350 Tyr Gln Asn Ile Thr Ile Gly Gly Gln Thr Pro Asp Gly Lys Asp Ala 355 360 365 Val Asn Pro Thr Ser Tyr Leu Ile Leu Arg Ala Ile Ala Gln Ala His 370 375 380 Leu Pro Gln Pro Asn Leu Thr Val Arg Tyr His His Gly Leu Ser Asp 385 390 395 400 Lys Phe Met Arg Glu Cys Val Glu Val Ile Lys Gln Gly Leu Gly Met 405 410 415 Pro Ala Phe Asn Asn Asp Glu Ile Ile Ile Pro Ser Phe Ile Arg Arg 420 425 430 Gly Val Lys Lys Glu Asp Ala Tyr Asn Tyr Ser Ala Ile Gly Cys Val 435 440 445 Glu Thr Ala Ile Pro Gly Lys Trp Gly Tyr Arg Cys Thr Gly Met Ser 450 455 460 Phe Ile Asn Phe Pro Arg Val Leu Leu Leu Ile Met Asn Gly Gly Ile 465 470 475 480 Asp Pro Glu Ser Gly Lys Arg Leu Leu Pro Asp Tyr Gly Lys Phe Thr 485 490 495 Asp Met Thr Ser Phe Asp Gln Leu Met Thr Ala Trp Asp Lys Ala Leu 500 505 510 Arg Glu Met Thr Arg Gln Ser Val Ile Ile Glu Asn Ser Cys Asp Leu 515 520 525 Ala Leu Glu Gln Asn Tyr Pro Asp Ile Leu Cys Ser Val Leu Thr Asp 530 535 540 Asp Cys Ile Gly Arg Gly Lys Thr Ile Lys Glu Gly Gly Ala Val Tyr 545 550 555 560 Asp Phe Ile Ser Gly Leu Gln Val Gly Ile Ala Asn Leu Ala Asp Ser 565 570 575 Leu Ala Ala Ile Lys Lys Leu Val Phe Glu Glu Lys Lys Leu Thr Thr 580 585 590 Thr Gln Leu Trp His Ala Leu Thr Thr Asp Phe Ala Asp Glu Asp Gly 595 600 605 Glu Lys Ile Arg Gln Met Leu Ile Asn Asp Ala Pro Lys Tyr Gly Asn 610 615 620 Asp Asp Asp Tyr Val Asp Asp Leu Ile Val Glu Ala Tyr Lys Pro Tyr 625 630 635 640 Ile Asp Glu Ile Ala Lys Tyr Lys Asn Thr Arg Tyr Gly Arg Gly Pro 645 650 655 Ile Gly Gly Leu Arg Tyr Ala Gly Thr Ser Ser Ile Ser Ala Asn Val 660 665 670 Gly Gln Gly His Ser Thr Leu Ala Thr Pro Asp Gly Arg His Ala Arg 675 680 685 Thr Pro Leu Ala Glu Gly Cys Ser Pro Glu His Ala Met Asp Thr Asp 690 695 700 Gly Pro Thr Ala Val Phe Lys Ser Val Ser Lys Leu Ser Thr Lys Asp 705 710 715 720 Ile Thr Gly Gly Val Leu Leu Asn Gln Lys Met Ser Pro Gln Ile Leu 725 730 735 Arg Ser Asp Glu Ser Cys Met Lys Leu Val Ala Leu Leu Arg Thr Phe 740 745 750 Phe Asn Arg Leu His Gly Tyr His Val Gln Tyr Asn Ile Val Ser Arg 755 760 765 Asp Thr Leu Ile Asp Ala Gln Asn His Pro Asp Lys His Arg Asp Leu 770 775 780 Ile Val Arg Val Ala Gly Tyr Ser Ala Phe Phe Val Gly Leu Ser Lys 785 790 795 800 Glu Thr Gln Asp Asp Ile Ile Glu Arg Thr Glu Gln Ser Leu 805 810 <210> SEQ ID NO 71 <211> LENGTH: 2259 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 71 atgattacat cagaaaagac aacaaaacca gcagcttgga aaggtttcaa aggcgggcac 60 tggcaggaag aaatcaacat tcgtgatttt attcaaaata acttcacaca gtacaatggc 120 gacgaaagct tcctggccgg accaacagcc gctactaaga ccttgaatga caaagtctta 180 gaattaaaga aacaagaacg tgccgctggt ggtgtgttgg atgctgatac taaagtcgtt 240 gcaacgatta cttcacacgg ccctggttat attcaaaaag atctcgaaaa gattgttggt 300 ctccagactg acaagccttt gaagcgggcc ttcatgccat ttggtggtat tcgaatggct 360 gatgacgctt tgaaatcata cggttatacc cctgatgaag aaaacgacaa gattttcact 420 gaatatcgca agactcataa ccaaggcgtc ttcgatgttt atactcctga catgcggaaa 480 gcacgtcact acaagatcat caccggacta ccagatgcat acgcacgtgg ccgtctcatt 540 cctgatcttc cacgggtcgc tgtttatggg atcgatcgtt taatggaaga caaagctaat 600 gactttgccc acattggtga tggtgaattg actgatgatg ttattcgcct ccgtgaagaa 660 gttcaagatc aataccgtgc tttagcagat atgaagaaga tggctgccag ttatggctac 720 gatattagca agcctgcaac taatgctcaa gaagctattc aatggatgta cttcgcttac 780 ttagctgcta tcaagaccca aaacggcgct gcaatgtccg ttggccggat tgatacaacg 840 atggacatct tcatccaacg tgacttggac aatggtgttc tggacgaaag ccaagctcaa 900 gaattaattg atcaattcgt catgaaacta cggatggttc ggttcatccg tactgaagaa 960 tacaattctc tcttctctgg tgacccaatc tgggcaacct tatcaatgtg tggtttaggc 1020 gtcgacggtc aacaccatgt gactaagact gctttccgga ttttaaagac tttggacaac 1080 atgggcgccg caccagaacc aaacatcacg attttatggt cagaccgctt accagaagac 1140 ttcaaacgtt acgcaactga agtttcaatc gacagttcaa ccattcagta tgaaaatgat 1200 gacttgatgc gggtacaatg gggtaccgat tattatggca ttgcttgctg tgtttccgca 1260 caaccaattg ctgatggaat ccagtacttc ggtgcccggg caaacttagc caaagcgatt 1320 ctttatgcca tcaatggtgg ccgcgacgaa attgctggag atcaagttgg ccctgcttac 1380 gaaccaatta cttcagaata catcgattac gacgaattca tgaagaaatt agacaagcaa 1440 atggattggt tagctgacac ttacgttaac tcactgaatg caattcatta tatgcatgat 1500 aagtactact atgaagctgc ccaattagct ttgaagaata ctgatcttga tcggaccttt 1560 gcaactggga tttctggctt atcacatgcc gcggattcaa tctcagctat caagtatggt 1620 cacgttaaag taattcgtga cgaacgtggt atcgccgttg acttcaaagc cgacaatgac 1680 tacccacgtt atgggaacaa tgacgatcgc gctgatgaca ttgctaaatg gttagtcaaa 1740 gaattataca gcaagatgaa cacgcatcac ctctatcgga atgccaaact ttcaacttct 1800 gttttgacga ttacctccaa cgttgtttat ggtaagaaca ctggtaccac gccaaatggc 1860 cgtcaaaaag gcgaaccatt ctcaccaggt gctaaccctg catacggtgc tgaaaagagt 1920 ggtgcattag cttcacttct ttcaactgcc aaattaccat accgttacgc aactgacggg 1980 atttccaaca cgttcggcgt tacccctaac acgttaggcc atgacctcga atcacggaaa 2040 gacacgttag taaacatgtt agacggttac atgaagaacg atgggatgca cttgaacatc 2100 aacgtcttca ataaagacac tttgattgat gctcagaaac accctgaaga atacccaaca 2160 ttaacggttc gggtttctgg ctattgtgtc tacttcgcag atttaaccaa ggaacaacaa 2220 gatgacgtta tttcacggac attcttcgaa tcaatgtaa 2259

<210> SEQ ID NO 72 <211> LENGTH: 752 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 72 Met Ile Thr Ser Glu Lys Thr Thr Lys Pro Ala Ala Trp Lys Gly Phe 1 5 10 15 Lys Gly Gly His Trp Gln Glu Glu Ile Asn Ile Arg Asp Phe Ile Gln 20 25 30 Asn Asn Phe Thr Gln Tyr Asn Gly Asp Glu Ser Phe Leu Ala Gly Pro 35 40 45 Thr Ala Ala Thr Lys Thr Leu Asn Asp Lys Val Leu Glu Leu Lys Lys 50 55 60 Gln Glu Arg Ala Ala Gly Gly Val Leu Asp Ala Asp Thr Lys Val Val 65 70 75 80 Ala Thr Ile Thr Ser His Gly Pro Gly Tyr Ile Gln Lys Asp Leu Glu 85 90 95 Lys Ile Val Gly Leu Gln Thr Asp Lys Pro Leu Lys Arg Ala Phe Met 100 105 110 Pro Phe Gly Gly Ile Arg Met Ala Asp Asp Ala Leu Lys Ser Tyr Gly 115 120 125 Tyr Thr Pro Asp Glu Glu Asn Asp Lys Ile Phe Thr Glu Tyr Arg Lys 130 135 140 Thr His Asn Gln Gly Val Phe Asp Val Tyr Thr Pro Asp Met Arg Lys 145 150 155 160 Ala Arg His Tyr Lys Ile Ile Thr Gly Leu Pro Asp Ala Tyr Ala Arg 165 170 175 Gly Arg Leu Ile Pro Asp Leu Pro Arg Val Ala Val Tyr Gly Ile Asp 180 185 190 Arg Leu Met Glu Asp Lys Ala Asn Asp Phe Ala His Ile Gly Asp Gly 195 200 205 Glu Leu Thr Asp Asp Val Ile Arg Leu Arg Glu Glu Val Gln Asp Gln 210 215 220 Tyr Arg Ala Leu Ala Asp Met Lys Lys Met Ala Ala Ser Tyr Gly Tyr 225 230 235 240 Asp Ile Ser Lys Pro Ala Thr Asn Ala Gln Glu Ala Ile Gln Trp Met 245 250 255 Tyr Phe Ala Tyr Leu Ala Ala Ile Lys Thr Gln Asn Gly Ala Ala Met 260 265 270 Ser Val Gly Arg Ile Asp Thr Thr Met Asp Ile Phe Ile Gln Arg Asp 275 280 285 Leu Asp Asn Gly Val Leu Asp Glu Ser Gln Ala Gln Glu Leu Ile Asp 290 295 300 Gln Phe Val Met Lys Leu Arg Met Val Arg Phe Ile Arg Thr Glu Glu 305 310 315 320 Tyr Asn Ser Leu Phe Ser Gly Asp Pro Ile Trp Ala Thr Leu Ser Met 325 330 335 Cys Gly Leu Gly Val Asp Gly Gln His His Val Thr Lys Thr Ala Phe 340 345 350 Arg Ile Leu Lys Thr Leu Asp Asn Met Gly Ala Ala Pro Glu Pro Asn 355 360 365 Ile Thr Ile Leu Trp Ser Asp Arg Leu Pro Glu Asp Phe Lys Arg Tyr 370 375 380 Ala Thr Glu Val Ser Ile Asp Ser Ser Thr Ile Gln Tyr Glu Asn Asp 385 390 395 400 Asp Leu Met Arg Val Gln Trp Gly Thr Asp Tyr Tyr Gly Ile Ala Cys 405 410 415 Cys Val Ser Ala Gln Pro Ile Ala Asp Gly Ile Gln Tyr Phe Gly Ala 420 425 430 Arg Ala Asn Leu Ala Lys Ala Ile Leu Tyr Ala Ile Asn Gly Gly Arg 435 440 445 Asp Glu Ile Ala Gly Asp Gln Val Gly Pro Ala Tyr Glu Pro Ile Thr 450 455 460 Ser Glu Tyr Ile Asp Tyr Asp Glu Phe Met Lys Lys Leu Asp Lys Gln 465 470 475 480 Met Asp Trp Leu Ala Asp Thr Tyr Val Asn Ser Leu Asn Ala Ile His 485 490 495 Tyr Met His Asp Lys Tyr Tyr Tyr Glu Ala Ala Gln Leu Ala Leu Lys 500 505 510 Asn Thr Asp Leu Asp Arg Thr Phe Ala Thr Gly Ile Ser Gly Leu Ser 515 520 525 His Ala Ala Asp Ser Ile Ser Ala Ile Lys Tyr Gly His Val Lys Val 530 535 540 Ile Arg Asp Glu Arg Gly Ile Ala Val Asp Phe Lys Ala Asp Asn Asp 545 550 555 560 Tyr Pro Arg Tyr Gly Asn Asn Asp Asp Arg Ala Asp Asp Ile Ala Lys 565 570 575 Trp Leu Val Lys Glu Leu Tyr Ser Lys Met Asn Thr His His Leu Tyr 580 585 590 Arg Asn Ala Lys Leu Ser Thr Ser Val Leu Thr Ile Thr Ser Asn Val 595 600 605 Val Tyr Gly Lys Asn Thr Gly Thr Thr Pro Asn Gly Arg Gln Lys Gly 610 615 620 Glu Pro Phe Ser Pro Gly Ala Asn Pro Ala Tyr Gly Ala Glu Lys Ser 625 630 635 640 Gly Ala Leu Ala Ser Leu Leu Ser Thr Ala Lys Leu Pro Tyr Arg Tyr 645 650 655 Ala Thr Asp Gly Ile Ser Asn Thr Phe Gly Val Thr Pro Asn Thr Leu 660 665 670 Gly His Asp Leu Glu Ser Arg Lys Asp Thr Leu Val Asn Met Leu Asp 675 680 685 Gly Tyr Met Lys Asn Asp Gly Met His Leu Asn Ile Asn Val Phe Asn 690 695 700 Lys Asp Thr Leu Ile Asp Ala Gln Lys His Pro Glu Glu Tyr Pro Thr 705 710 715 720 Leu Thr Val Arg Val Ser Gly Tyr Cys Val Tyr Phe Ala Asp Leu Thr 725 730 735 Lys Glu Gln Gln Asp Asp Val Ile Ser Arg Thr Phe Phe Glu Ser Met 740 745 750 <210> SEQ ID NO 73 <211> LENGTH: 804 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 73 atgccaacga tcacaactaa gacgcccgta aaaggactaa tatttaacat tcaaaaattt 60 agtatcaatg atggaccagg tattcgaaca gtagttttct ttaaagggtg cccgttacgc 120 tgcaagtggt gttctaatcc agaatcacaa tcaggtgagc aagaatcaat gtatgatgaa 180 cagaccgcca agcaaaccat cgtcggtgat tatatgacgg ttgatgatat tatgaaagtt 240 attctacaag ataaagactt ctatgaagag tctggcggtg gggtaacctt ctctggtggt 300 gaagttcttt ttcaagcttc ctttgcgatt gagcttgcta aggcagttaa agcagctggc 360 attaatttag cctgtgagac aactggttac gcacggccta aggtttttaa tgaattcatg 420 tcttatatgg acttcatgta ttatgactgt aaacaatggg acccagccca acatcgaatc 480 ggaacgggtg ccgataacgg ggtaatttta cgtaacttag caactgcagt gcaagctcat 540 caaaagatga tggttcggat tccggttatt ccaggtttta attatacatt gaatgacgcg 600 gatcattttg gacaactatt taatcagatt ggcgtaaccg aagttgaatt attgccattt 660 caccagtttg ggttgaaaaa gtatcaagat ttgggccgaa aatatgcgct agttaatgtt 720 aaacagttac aagcggatga cttaattgat tatgctgaac atattcgtgc acatggtgtt 780 aaagtacggg tgaatgggtg gtaa 804 <210> SEQ ID NO 74 <211> LENGTH: 267 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 74 Met Pro Thr Ile Thr Thr Lys Thr Pro Val Lys Gly Leu Ile Phe Asn 1 5 10 15 Ile Gln Lys Phe Ser Ile Asn Asp Gly Pro Gly Ile Arg Thr Val Val 20 25 30 Phe Phe Lys Gly Cys Pro Leu Arg Cys Lys Trp Cys Ser Asn Pro Glu 35 40 45 Ser Gln Ser Gly Glu Gln Glu Ser Met Tyr Asp Glu Gln Thr Ala Lys 50 55 60 Gln Thr Ile Val Gly Asp Tyr Met Thr Val Asp Asp Ile Met Lys Val 65 70 75 80 Ile Leu Gln Asp Lys Asp Phe Tyr Glu Glu Ser Gly Gly Gly Val Thr 85 90 95 Phe Ser Gly Gly Glu Val Leu Phe Gln Ala Ser Phe Ala Ile Glu Leu 100 105 110 Ala Lys Ala Val Lys Ala Ala Gly Ile Asn Leu Ala Cys Glu Thr Thr 115 120 125 Gly Tyr Ala Arg Pro Lys Val Phe Asn Glu Phe Met Ser Tyr Met Asp 130 135 140 Phe Met Tyr Tyr Asp Cys Lys Gln Trp Asp Pro Ala Gln His Arg Ile 145 150 155 160 Gly Thr Gly Ala Asp Asn Gly Val Ile Leu Arg Asn Leu Ala Thr Ala 165 170 175 Val Gln Ala His Gln Lys Met Met Val Arg Ile Pro Val Ile Pro Gly 180 185 190 Phe Asn Tyr Thr Leu Asn Asp Ala Asp His Phe Gly Gln Leu Phe Asn 195 200 205 Gln Ile Gly Val Thr Glu Val Glu Leu Leu Pro Phe His Gln Phe Gly 210 215 220 Leu Lys Lys Tyr Gln Asp Leu Gly Arg Lys Tyr Ala Leu Val Asn Val 225 230 235 240 Lys Gln Leu Gln Ala Asp Asp Leu Ile Asp Tyr Ala Glu His Ile Arg 245 250 255 Ala His Gly Val Lys Val Arg Val Asn Gly Trp 260 265 <210> SEQ ID NO 75 <211> LENGTH: 822 <212> TYPE: DNA <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 75

atggaaaaca aacaagtttc aacaacgcaa gcggcggcaa aggagccttt gataggctac 60 gttcactcca tcgaaacgtt tggctccgtt gacggaccag gtatccgtta cgtggcattc 120 cttcaaggat gccacatgcg ttgccaatac tgtcacaacc ctgatacttg gaaactcaac 180 gttggcgatc aaatgacggc cgacgagatt ctcgaagacg cggctaaata ccgggctttc 240 tggggcaaga cgggtggcat cacagtcagt ggtggtgaat cactggtaca aatcgacttc 300 atcttagact tattcgaaaa agccaaggcg atgaatatca gtacttgtct ggatacctct 360 ggacagcctt ttacccgaga acaacctttc tttgacaagt tcgaacgtct aatgaaggtc 420 acggacattt cgttggtcga cattaagcac atcgattctg ccaaacacaa gcagttgacc 480 cagtatggga acgaaaatat cttggatatg attcagtaca tggcccaaca ccacgatgat 540 atgtggattc gtcacgtcct ggttccccaa cggactgatt acgatgaaga cttgaagaaa 600 ctcggcgatt acattgctaa aattccaaac gacgtcgttc aaaaagtcga agtattgccg 660 taccatactt tgggcgttaa aaaatatcat gaaatgaaga tcaagtaccg gcttgaagga 720 atcgagtctc caacccaaga tcgggtggca aatgccgaaa agctactgca cactgctgat 780 tacaacgggt acaagacatg gatgccattg ccaaaacttt aa 822 <210> SEQ ID NO 76 <211> LENGTH: 273 <212> TYPE: PRT <213> ORGANISM: Lactobacillus plantarum <400> SEQUENCE: 76 Met Glu Asn Lys Gln Val Ser Thr Thr Gln Ala Ala Ala Lys Glu Pro 1 5 10 15 Leu Ile Gly Tyr Val His Ser Ile Glu Thr Phe Gly Ser Val Asp Gly 20 25 30 Pro Gly Ile Arg Tyr Val Ala Phe Leu Gln Gly Cys His Met Arg Cys 35 40 45 Gln Tyr Cys His Asn Pro Asp Thr Trp Lys Leu Asn Val Gly Asp Gln 50 55 60 Met Thr Ala Asp Glu Ile Leu Glu Asp Ala Ala Lys Tyr Arg Ala Phe 65 70 75 80 Trp Gly Lys Thr Gly Gly Ile Thr Val Ser Gly Gly Glu Ser Leu Val 85 90 95 Gln Ile Asp Phe Ile Leu Asp Leu Phe Glu Lys Ala Lys Ala Met Asn 100 105 110 Ile Ser Thr Cys Leu Asp Thr Ser Gly Gln Pro Phe Thr Arg Glu Gln 115 120 125 Pro Phe Phe Asp Lys Phe Glu Arg Leu Met Lys Val Thr Asp Ile Ser 130 135 140 Leu Val Asp Ile Lys His Ile Asp Ser Ala Lys His Lys Gln Leu Thr 145 150 155 160 Gln Tyr Gly Asn Glu Asn Ile Leu Asp Met Ile Gln Tyr Met Ala Gln 165 170 175 His His Asp Asp Met Trp Ile Arg His Val Leu Val Pro Gln Arg Thr 180 185 190 Asp Tyr Asp Glu Asp Leu Lys Lys Leu Gly Asp Tyr Ile Ala Lys Ile 195 200 205 Pro Asn Asp Val Val Gln Lys Val Glu Val Leu Pro Tyr His Thr Leu 210 215 220 Gly Val Lys Lys Tyr His Glu Met Lys Ile Lys Tyr Arg Leu Glu Gly 225 230 235 240 Ile Glu Ser Pro Thr Gln Asp Arg Val Ala Asn Ala Glu Lys Leu Leu 245 250 255 His Thr Ala Asp Tyr Asn Gly Tyr Lys Thr Trp Met Pro Leu Pro Lys 260 265 270 Leu <210> SEQ ID NO 77 <211> LENGTH: 2364 <212> TYPE: DNA <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 77 atgaaaaccg aagttacgga aaatatcttt gaacaagctt gggatggttt taaaggaacc 60 aactggcgcg ataaagcaag cgttactcgc tttgtacaag aaaactacaa accatatgat 120 ggtgatgaaa gctttcttgc tgggccaaca gaacgtacac ttaaagtaaa gaaaattatt 180 gaagatacaa aaaatcacta cgaagaagta ggatttccct tcgatactga ccgcgtaacc 240 tctattgata aaatccctgc tggatatatc gatgctaatg ataaagaact tgaactcatc 300 tatgggatgc aaaatagcga acttttccgc ttgaatttca tgccaagagg tggacttcgt 360 gttgctgaaa agattttgac agaacacggt ctctcagttg acccaggctt gcatgatgtt 420 ttgtcacaaa caatgacttc tgtaaatgat ggaatctttc gtgcttatac ttcagcaatt 480 cgtaaagcac gtcatgctca tactgtaaca ggtttgccag atgcttactc tcgtggacgt 540 atcattggtg tctatgcacg tcttgccctt tacggtgctg attaccttat gaaggaaaaa 600 gcaaaagaat gggatgcaat cactgaaatt aacgaagaaa acattcgtct taaagaagaa 660 attaatatgc aataccaagc tttgcaagaa gttgtaaact ttggtgcttt atatggtctt 720 gatgtttcac gtccagctat gaacgtaaaa gaagcaatcc aatgggttaa catcgcttat 780 atggcagtat gtcgtgtcat taatggagct gcaacttcac ttggacgtgt tccaatcgtt 840 cttgatatct ttgcagaacg tgaccttgct cgtggaacat ttactgaaca agaaattcaa 900 gaatttgttg atgatttcgt tttgaagctt cgtacaatga aatttgcgcg tgcagctgct 960 tatgatgaac tttattctgg tgacccaaca ttcatcacaa catctatggc tggtatgggt 1020 aatgacggac gtcaccgtgt cactaaaatg gactaccgtt tcttgaacac acttgataca 1080 atcggaaatg ctccagaacc aaacttgaca gtcctttggg attctaaact tccttactca 1140 ttcaaacgtt attcaatgtc tatgagccac aagcattctt ctattcaata tgaaggtgtt 1200 gaaacaatgg ctaaagatgg atatggcgaa atgtcatgta tctcttgttg tgtctcacca 1260 cttgatccag aaaatgaaga aggacgtcat aacctccaat actttggtgc gcgtgtaaac 1320 gtcttgaaag caatgttgac tggtttgaac ggtggttatg atgacgttca taaagattat 1380 aaagtattcg acatcgaacc tgttcgtgac gaaattcttg actatgatac agttatggaa 1440 aactttgaca aatctctcga ctggttgact gatacttatg ttgatgcaat gaatatcatt 1500 cattacatga ctgataaata taactatgaa gcagttcaaa tggccttctt gcctactaaa 1560 gttcgtgcta acatgggatt tggtatctgt ggattcgcaa atacagttga ttcactttca 1620 gcaattaaat atgctaaagt taaaacattg cgtgatgaaa atggctatat ctacgattac 1680 gaagtagaag gtgatttccc tcgttatggt gaagatgatg atcgtgctga tgatattgct 1740 aaacttgtca tgaaaatgta ccatgaaaaa ttagcttcac acaaacttta caaaaatgct 1800 gaagctactg tttcactttt gacaattaca tctaacgttg cttactctaa acaaactggt 1860 aattctccag tacataaagg agtattcctc aatgaagatg gtacagtaaa taaatctaaa 1920 cttgaattct tctcaccagg tgctaaccca tctaataaag ctaagggtgg ttggttgcaa 1980 aatcttcgct cattggctaa gttggaattc aaagatgcaa atgatggtat ttcattgact 2040 actcaagttt cacctcgtgc acttggtaaa actcgtgatg aacaagtgga taacttggtt 2100 caaattcttg atggatactt cacaccaggt gctttgatta atggtactga atttgcaggt 2160 caacacgtta acttgaacgt aatggacctt aaagatgttt acgataaaat catgcgtggt 2220 gaagatgtta tcgttcgtat ctctggttac tgtgtcaata ctaaatacct cacaccagaa 2280 caaaaacaag aattaactga acgtgtcttc catgaagttc tttcaaacga tgatgaagaa 2340 gtaatgcata cttcaaacat ctaa 2364 <210> SEQ ID NO 78 <211> LENGTH: 787 <212> TYPE: PRT <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 78 Met Lys Thr Glu Val Thr Glu Asn Ile Phe Glu Gln Ala Trp Asp Gly 1 5 10 15 Phe Lys Gly Thr Asn Trp Arg Asp Lys Ala Ser Val Thr Arg Phe Val 20 25 30 Gln Glu Asn Tyr Lys Pro Tyr Asp Gly Asp Glu Ser Phe Leu Ala Gly 35 40 45 Pro Thr Glu Arg Thr Leu Lys Val Lys Lys Ile Ile Glu Asp Thr Lys 50 55 60 Asn His Tyr Glu Glu Val Gly Phe Pro Phe Asp Thr Asp Arg Val Thr 65 70 75 80 Ser Ile Asp Lys Ile Pro Ala Gly Tyr Ile Asp Ala Asn Asp Lys Glu 85 90 95 Leu Glu Leu Ile Tyr Gly Met Gln Asn Ser Glu Leu Phe Arg Leu Asn 100 105 110 Phe Met Pro Arg Gly Gly Leu Arg Val Ala Glu Lys Ile Leu Thr Glu 115 120 125 His Gly Leu Ser Val Asp Pro Gly Leu His Asp Val Leu Ser Gln Thr 130 135 140 Met Thr Ser Val Asn Asp Gly Ile Phe Arg Ala Tyr Thr Ser Ala Ile 145 150 155 160 Arg Lys Ala Arg His Ala His Thr Val Thr Gly Leu Pro Asp Ala Tyr 165 170 175 Ser Arg Gly Arg Ile Ile Gly Val Tyr Ala Arg Leu Ala Leu Tyr Gly 180 185 190 Ala Asp Tyr Leu Met Lys Glu Lys Ala Lys Glu Trp Asp Ala Ile Thr 195 200 205 Glu Ile Asn Glu Glu Asn Ile Arg Leu Lys Glu Glu Ile Asn Met Gln 210 215 220 Tyr Gln Ala Leu Gln Glu Val Val Asn Phe Gly Ala Leu Tyr Gly Leu 225 230 235 240 Asp Val Ser Arg Pro Ala Met Asn Val Lys Glu Ala Ile Gln Trp Val 245 250 255 Asn Ile Ala Tyr Met Ala Val Cys Arg Val Ile Asn Gly Ala Ala Thr 260 265 270 Ser Leu Gly Arg Val Pro Ile Val Leu Asp Ile Phe Ala Glu Arg Asp 275 280 285 Leu Ala Arg Gly Thr Phe Thr Glu Gln Glu Ile Gln Glu Phe Val Asp 290 295 300 Asp Phe Val Leu Lys Leu Arg Thr Met Lys Phe Ala Arg Ala Ala Ala 305 310 315 320 Tyr Asp Glu Leu Tyr Ser Gly Asp Pro Thr Phe Ile Thr Thr Ser Met 325 330 335 Ala Gly Met Gly Asn Asp Gly Arg His Arg Val Thr Lys Met Asp Tyr 340 345 350

Arg Phe Leu Asn Thr Leu Asp Thr Ile Gly Asn Ala Pro Glu Pro Asn 355 360 365 Leu Thr Val Leu Trp Asp Ser Lys Leu Pro Tyr Ser Phe Lys Arg Tyr 370 375 380 Ser Met Ser Met Ser His Lys His Ser Ser Ile Gln Tyr Glu Gly Val 385 390 395 400 Glu Thr Met Ala Lys Asp Gly Tyr Gly Glu Met Ser Cys Ile Ser Cys 405 410 415 Cys Val Ser Pro Leu Asp Pro Glu Asn Glu Glu Gly Arg His Asn Leu 420 425 430 Gln Tyr Phe Gly Ala Arg Val Asn Val Leu Lys Ala Met Leu Thr Gly 435 440 445 Leu Asn Gly Gly Tyr Asp Asp Val His Lys Asp Tyr Lys Val Phe Asp 450 455 460 Ile Glu Pro Val Arg Asp Glu Ile Leu Asp Tyr Asp Thr Val Met Glu 465 470 475 480 Asn Phe Asp Lys Ser Leu Asp Trp Leu Thr Asp Thr Tyr Val Asp Ala 485 490 495 Met Asn Ile Ile His Tyr Met Thr Asp Lys Tyr Asn Tyr Glu Ala Val 500 505 510 Gln Met Ala Phe Leu Pro Thr Lys Val Arg Ala Asn Met Gly Phe Gly 515 520 525 Ile Cys Gly Phe Ala Asn Thr Val Asp Ser Leu Ser Ala Ile Lys Tyr 530 535 540 Ala Lys Val Lys Thr Leu Arg Asp Glu Asn Gly Tyr Ile Tyr Asp Tyr 545 550 555 560 Glu Val Glu Gly Asp Phe Pro Arg Tyr Gly Glu Asp Asp Asp Arg Ala 565 570 575 Asp Asp Ile Ala Lys Leu Val Met Lys Met Tyr His Glu Lys Leu Ala 580 585 590 Ser His Lys Leu Tyr Lys Asn Ala Glu Ala Thr Val Ser Leu Leu Thr 595 600 605 Ile Thr Ser Asn Val Ala Tyr Ser Lys Gln Thr Gly Asn Ser Pro Val 610 615 620 His Lys Gly Val Phe Leu Asn Glu Asp Gly Thr Val Asn Lys Ser Lys 625 630 635 640 Leu Glu Phe Phe Ser Pro Gly Ala Asn Pro Ser Asn Lys Ala Lys Gly 645 650 655 Gly Trp Leu Gln Asn Leu Arg Ser Leu Ala Lys Leu Glu Phe Lys Asp 660 665 670 Ala Asn Asp Gly Ile Ser Leu Thr Thr Gln Val Ser Pro Arg Ala Leu 675 680 685 Gly Lys Thr Arg Asp Glu Gln Val Asp Asn Leu Val Gln Ile Leu Asp 690 695 700 Gly Tyr Phe Thr Pro Gly Ala Leu Ile Asn Gly Thr Glu Phe Ala Gly 705 710 715 720 Gln His Val Asn Leu Asn Val Met Asp Leu Lys Asp Val Tyr Asp Lys 725 730 735 Ile Met Arg Gly Glu Asp Val Ile Val Arg Ile Ser Gly Tyr Cys Val 740 745 750 Asn Thr Lys Tyr Leu Thr Pro Glu Gln Lys Gln Glu Leu Thr Glu Arg 755 760 765 Val Phe His Glu Val Leu Ser Asn Asp Asp Glu Glu Val Met His Thr 770 775 780 Ser Asn Ile 785 <210> SEQ ID NO 79 <211> LENGTH: 795 <212> TYPE: DNA <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 79 atgatgtcag agaatataga tgaacttaaa aaagttactg gactgattca ttcaactgaa 60 tcttttggtt ctgttgatgg ccctggggtc cgttttatta ttttcatgca aggctgtcgg 120 atgcgttgca aatattgtca caaccctgat acttgggcat taaagtcaga taaagcgaca 180 gagcgtaccg tagaagatgt catggatgag gcacttcgtt ttagaggttt ttggggagag 240 aaaggtggaa ttaccgtttc tggtggtgag gcgctccttc aaattgactt tgtattagct 300 cttttcaaat atgcaaaatc tctcggtatt catacaacac ttgatacagc ggctcaacca 360 tatttgactg ataaatatgt aaccgaaaaa attgatgagt tactagatta taccgactta 420 gtattattag acattaaaga aataaatcca gaacgacaca aagaattgac agctaataaa 480 aacgataata ttttagcttt tgcacagtat ttatcagacc gtggtaatgc aatgtgggtt 540 cgtcacgttc ttgttcctgg tgaaagtgat tttgatgaag atttagttca attaggtgaa 600 tttgtaaaaa ctttaaaaaa tgtcttgaaa tttgaaattt taccctacca tacaatgggt 660 gaatttaaat ggcgtgaatt aggttggaaa tatccgcttg aaggtgtgaa acctccaaca 720 aaagatcgtg ttcataatgc taaagaaatc atgaatacag aatcttatca agattactta 780 gaacgtataa gataa 795 <210> SEQ ID NO 80 <211> LENGTH: 264 <212> TYPE: PRT <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 80 Met Met Ser Glu Asn Ile Asp Glu Leu Lys Lys Val Thr Gly Leu Ile 1 5 10 15 His Ser Thr Glu Ser Phe Gly Ser Val Asp Gly Pro Gly Val Arg Phe 20 25 30 Ile Ile Phe Met Gln Gly Cys Arg Met Arg Cys Lys Tyr Cys His Asn 35 40 45 Pro Asp Thr Trp Ala Leu Lys Ser Asp Lys Ala Thr Glu Arg Thr Val 50 55 60 Glu Asp Val Met Asp Glu Ala Leu Arg Phe Arg Gly Phe Trp Gly Glu 65 70 75 80 Lys Gly Gly Ile Thr Val Ser Gly Gly Glu Ala Leu Leu Gln Ile Asp 85 90 95 Phe Val Leu Ala Leu Phe Lys Tyr Ala Lys Ser Leu Gly Ile His Thr 100 105 110 Thr Leu Asp Thr Ala Ala Gln Pro Tyr Leu Thr Asp Lys Tyr Val Thr 115 120 125 Glu Lys Ile Asp Glu Leu Leu Asp Tyr Thr Asp Leu Val Leu Leu Asp 130 135 140 Ile Lys Glu Ile Asn Pro Glu Arg His Lys Glu Leu Thr Ala Asn Lys 145 150 155 160 Asn Asp Asn Ile Leu Ala Phe Ala Gln Tyr Leu Ser Asp Arg Gly Asn 165 170 175 Ala Met Trp Val Arg His Val Leu Val Pro Gly Glu Ser Asp Phe Asp 180 185 190 Glu Asp Leu Val Gln Leu Gly Glu Phe Val Lys Thr Leu Lys Asn Val 195 200 205 Leu Lys Phe Glu Ile Leu Pro Tyr His Thr Met Gly Glu Phe Lys Trp 210 215 220 Arg Glu Leu Gly Trp Lys Tyr Pro Leu Glu Gly Val Lys Pro Pro Thr 225 230 235 240 Lys Asp Arg Val His Asn Ala Lys Glu Ile Met Asn Thr Glu Ser Tyr 245 250 255 Gln Asp Tyr Leu Glu Arg Ile Arg 260 <210> SEQ ID NO 81 <211> LENGTH: 2310 <212> TYPE: DNA <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 81 atggcaacgg ttaaaactaa cacagatgtt tttgaaaaag cgtgggaagg ctttaaagga 60 actgactgga aagaaaaagc aagtgtgtct cgcttcgtac aagcaaacta cacaccatat 120 gatggtgatg aaagcttcct tgcaggacca actgaacgct cacttaaaat caaaaaaatc 180 attgaagaaa ctaaagctca ctacgaagaa actcgtttcc caatggatac tcgtccgaca 240 tcaatcgcag atattcctgc cggctatatt tcaaaagacg acgaactaat ctacggtatt 300 caaaatgatg agttattcaa attgaatttc atgccaaaag gcggaattcg tatggcagaa 360 acagctctca aggaacatgg ctatgaacct gatccagctg ttcacgaaat ttttacaaaa 420 catgtaacta cagtaaatga cggtatcttc cgtgcttata catcaaatat ccgtcgtgca 480 cgtcacgcac acactataac tggacttcca gatgcttact ctcgtggacg tatcatcggt 540 gtttatgctc gccttgctct ttacggtgct gacttcttga tgcaagaaaa agtaaacgac 600 tggaactcta tcgaagaaat caacgaagaa actattcgtc ttcgtgaaga agttaacctt 660 caataccaag cacttcaaga tgttgttcgc cttggtgacc tttacggtgt agatgttcgt 720 cgtccagcct tcgatactaa agaagctatc caatggacaa acattgcttt tatggctgta 780 tgtcgtgtta tcaatggtgc ggctacttca cttggtcgtg tgccaatcgt ccttgacata 840 tatgcagaac gtgaccttgc tcgtggtact tacactgaat cagaaatcca agaattcgtt 900 gatgattttg tcttgaaact tcgtactgta aaattcgcac gtacaaaagc ttacgacgaa 960 ctttactcag gtgacccaac attcatcaca acttctatgg ctggtatggg tgctgacgga 1020 cgtcaccgtg ttactaaaat ggactaccgt ttcttgaaca cacttgataa tattggtaat 1080 gctccagaac caaacttgac agttctttgg tctgacaaat tgccttactc attccgtcgc 1140 tactgtatgc acatgagtca caagcactct tctattcaat acgaaggtgt gactactatg 1200 gctaaagacg gatacggtga aatgagctgt atctcatgtt gtgtatcacc acttgaccca 1260 gaaaacgaag aacaacgcca caacatccaa tacttcggtg ctcgtgttaa cgtacttaaa 1320 gcccttctta ctggtttgaa cggtggttac gacgatgttc ataaagacta caaagtattt 1380 gacatcgatc cagtccgtga tgaagttctt gactttgaca ctgttaaagc taacttcgaa 1440 aaatctcttg actggttgac tgacacttat gtagatgccc ttaacatcat ccactacatg 1500 actgataagt acaactacga agctgttcaa atggccttct tgccaactaa acaacgtgct 1560 aacatgggat tcggtatctg tggtttcgca aatactgttg atacattgtc agctatcaag 1620 tacgctacag ttaaaccaat ccgtgacgaa gatggctaca tctacgacta cgaaacaatc 1680 ggtgaatacc cacgttgggg tgaagatgac ccacgttcaa acgaattggc agaatggttg 1740 attgaagctt acactactcg tcttcgtagc cataaactct acaaagatgc agaagctaca 1800 gtttcacttc ttacaatcac ttcgaacgtt gcttactcta aacaaactgg taactctcca 1860 gttcacaaag gggtatacct caacgaagat ggttcagtga acttgtctaa attggaattc 1920

ttctcaccag gtgctaaccc atctaacaaa gctaaaggtg gatggttgca aaacttgaac 1980 tcacttgcaa gccttgactt cggttatgca gctgacggta tctcacttac tactcaagta 2040 tcacctcgtg cccttggtaa gactcgcgac gaacaagttg ataacctcgt aactatcctt 2100 gacggatact tcgaaaacgg tggacaacac cttaacttga acgttatgga cttgtcagct 2160 gtttacaaaa agatcatgag cggtgaagat gttatcgtac gtatctctgg atactgtgta 2220 aacactaaat acctcactcc agaacaaaaa actgaattga cacaacgtgt cttccacgaa 2280 gttctttcaa cggacgatgc tatgggataa 2310 <210> SEQ ID NO 82 <211> LENGTH: 769 <212> TYPE: PRT <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 82 Met Ala Thr Val Lys Thr Asn Thr Asp Val Phe Glu Lys Ala Trp Glu 1 5 10 15 Gly Phe Lys Gly Thr Asp Trp Lys Glu Lys Ala Ser Val Ser Arg Phe 20 25 30 Val Gln Ala Asn Tyr Thr Pro Tyr Asp Gly Asp Glu Ser Phe Leu Ala 35 40 45 Gly Pro Thr Glu Arg Ser Leu Lys Ile Lys Lys Ile Ile Glu Glu Thr 50 55 60 Lys Ala His Tyr Glu Glu Thr Arg Phe Pro Met Asp Thr Arg Pro Thr 65 70 75 80 Ser Ile Ala Asp Ile Pro Ala Gly Tyr Ile Ser Lys Asp Asp Glu Leu 85 90 95 Ile Tyr Gly Ile Gln Asn Asp Glu Leu Phe Lys Leu Asn Phe Met Pro 100 105 110 Lys Gly Gly Ile Arg Met Ala Glu Thr Ala Leu Lys Glu His Gly Tyr 115 120 125 Glu Pro Asp Pro Ala Val His Glu Ile Phe Thr Lys His Val Thr Thr 130 135 140 Val Asn Asp Gly Ile Phe Arg Ala Tyr Thr Ser Asn Ile Arg Arg Ala 145 150 155 160 Arg His Ala His Thr Ile Thr Gly Leu Pro Asp Ala Tyr Ser Arg Gly 165 170 175 Arg Ile Ile Gly Val Tyr Ala Arg Leu Ala Leu Tyr Gly Ala Asp Phe 180 185 190 Leu Met Gln Glu Lys Val Asn Asp Trp Asn Ser Ile Glu Glu Ile Asn 195 200 205 Glu Glu Thr Ile Arg Leu Arg Glu Glu Val Asn Leu Gln Tyr Gln Ala 210 215 220 Leu Gln Asp Val Val Arg Leu Gly Asp Leu Tyr Gly Val Asp Val Arg 225 230 235 240 Arg Pro Ala Phe Asp Thr Lys Glu Ala Ile Gln Trp Thr Asn Ile Ala 245 250 255 Phe Met Ala Val Cys Arg Val Ile Asn Gly Ala Ala Thr Ser Leu Gly 260 265 270 Arg Val Pro Ile Val Leu Asp Ile Tyr Ala Glu Arg Asp Leu Ala Arg 275 280 285 Gly Thr Tyr Thr Glu Ser Glu Ile Gln Glu Phe Val Asp Asp Phe Val 290 295 300 Leu Lys Leu Arg Thr Val Lys Phe Ala Arg Thr Lys Ala Tyr Asp Glu 305 310 315 320 Leu Tyr Ser Gly Asp Pro Thr Phe Ile Thr Thr Ser Met Ala Gly Met 325 330 335 Gly Ala Asp Gly Arg His Arg Val Thr Lys Met Asp Tyr Arg Phe Leu 340 345 350 Asn Thr Leu Asp Asn Ile Gly Asn Ala Pro Glu Pro Asn Leu Thr Val 355 360 365 Leu Trp Ser Asp Lys Leu Pro Tyr Ser Phe Arg Arg Tyr Cys Met His 370 375 380 Met Ser His Lys His Ser Ser Ile Gln Tyr Glu Gly Val Thr Thr Met 385 390 395 400 Ala Lys Asp Gly Tyr Gly Glu Met Ser Cys Ile Ser Cys Cys Val Ser 405 410 415 Pro Leu Asp Pro Glu Asn Glu Glu Gln Arg His Asn Ile Gln Tyr Phe 420 425 430 Gly Ala Arg Val Asn Val Leu Lys Ala Leu Leu Thr Gly Leu Asn Gly 435 440 445 Gly Tyr Asp Asp Val His Lys Asp Tyr Lys Val Phe Asp Ile Asp Pro 450 455 460 Val Arg Asp Glu Val Leu Asp Phe Asp Thr Val Lys Ala Asn Phe Glu 465 470 475 480 Lys Ser Leu Asp Trp Leu Thr Asp Thr Tyr Val Asp Ala Leu Asn Ile 485 490 495 Ile His Tyr Met Thr Asp Lys Tyr Asn Tyr Glu Ala Val Gln Met Ala 500 505 510 Phe Leu Pro Thr Lys Gln Arg Ala Asn Met Gly Phe Gly Ile Cys Gly 515 520 525 Phe Ala Asn Thr Val Asp Thr Leu Ser Ala Ile Lys Tyr Ala Thr Val 530 535 540 Lys Pro Ile Arg Asp Glu Asp Gly Tyr Ile Tyr Asp Tyr Glu Thr Ile 545 550 555 560 Gly Glu Tyr Pro Arg Trp Gly Glu Asp Asp Pro Arg Ser Asn Glu Leu 565 570 575 Ala Glu Trp Leu Ile Glu Ala Tyr Thr Thr Arg Leu Arg Ser His Lys 580 585 590 Leu Tyr Lys Asp Ala Glu Ala Thr Val Ser Leu Leu Thr Ile Thr Ser 595 600 605 Asn Val Ala Tyr Ser Lys Gln Thr Gly Asn Ser Pro Val His Lys Gly 610 615 620 Val Tyr Leu Asn Glu Asp Gly Ser Val Asn Leu Ser Lys Leu Glu Phe 625 630 635 640 Phe Ser Pro Gly Ala Asn Pro Ser Asn Lys Ala Lys Gly Gly Trp Leu 645 650 655 Gln Asn Leu Asn Ser Leu Ala Ser Leu Asp Phe Gly Tyr Ala Ala Asp 660 665 670 Gly Ile Ser Leu Thr Thr Gln Val Ser Pro Arg Ala Leu Gly Lys Thr 675 680 685 Arg Asp Glu Gln Val Asp Asn Leu Val Thr Ile Leu Asp Gly Tyr Phe 690 695 700 Glu Asn Gly Gly Gln His Leu Asn Leu Asn Val Met Asp Leu Ser Ala 705 710 715 720 Val Tyr Lys Lys Ile Met Ser Gly Glu Asp Val Ile Val Arg Ile Ser 725 730 735 Gly Tyr Cys Val Asn Thr Lys Tyr Leu Thr Pro Glu Gln Lys Thr Glu 740 745 750 Leu Thr Gln Arg Val Phe His Glu Val Leu Ser Thr Asp Asp Ala Met 755 760 765 Gly <210> SEQ ID NO 83 <211> LENGTH: 801 <212> TYPE: DNA <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 83 atggcagaaa ttgattacag tcaggtgact ggacttgttc attcaaccga aagtttcgga 60 tccgtagatg gtcctggtat ccgttttatt gtgtttatgc aaggctgtaa gctgcgttgc 120 caatattgtc ataacccaga tacttgggcc atgaagtcaa ataaggctgt tgaacgtaca 180 gttgaagatg tcttagaaga ggctcttcgc ttccgtcatt tctggggtga gcatggtgga 240 atcactgtat caggtggtga agccatgctt cagattgatt ttgtcactgc cctctttaca 300 gaggctaaga agttagggat tcactgtacg cttgatacgt gtggcttgtc ttatcgtaat 360 actccagagt atcatgaagt tgtcgacaaa cttttggctg taactgactt ggttctactg 420 gatatcaaag agattgaccc cgaacaacac aagtttgtga cccgtcaacc taataagaat 480 atcttggaat ttgctcaata tctgtctgat aaacaagttc cggtctggat tcgtcatgtc 540 ttggtacctg gtttgacaga ttttgacgaa cacttggtta agctcggcga gtttgtaaag 600 accttgaaaa atgtcgataa atttgaaatt cttccatatc atacgatggg ggaattcaag 660 tggcgtgaac ttggcatccc ttatccattg gaaggtgtca aaccaccaac tgcagatcgt 720 gttaaaaatg ctaaggctct tatgcatacg gaaacttatc aagagtataa gaatcgtatc 780 ggggttaaaa ccttggatta a 801 <210> SEQ ID NO 84 <211> LENGTH: 266 <212> TYPE: PRT <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 84 Met Ala Glu Ile Asp Tyr Ser Gln Val Thr Gly Leu Val His Ser Thr 1 5 10 15 Glu Ser Phe Gly Ser Val Asp Gly Pro Gly Ile Arg Phe Ile Val Phe 20 25 30 Met Gln Gly Cys Lys Leu Arg Cys Gln Tyr Cys His Asn Pro Asp Thr 35 40 45 Trp Ala Met Lys Ser Asn Lys Ala Val Glu Arg Thr Val Glu Asp Val 50 55 60 Leu Glu Glu Ala Leu Arg Phe Arg His Phe Trp Gly Glu His Gly Gly 65 70 75 80 Ile Thr Val Ser Gly Gly Glu Ala Met Leu Gln Ile Asp Phe Val Thr 85 90 95 Ala Leu Phe Thr Glu Ala Lys Lys Leu Gly Ile His Cys Thr Leu Asp 100 105 110 Thr Cys Gly Leu Ser Tyr Arg Asn Thr Pro Glu Tyr His Glu Val Val 115 120 125 Asp Lys Leu Leu Ala Val Thr Asp Leu Val Leu Leu Asp Ile Lys Glu 130 135 140 Ile Asp Pro Glu Gln His Lys Phe Val Thr Arg Gln Pro Asn Lys Asn 145 150 155 160 Ile Leu Glu Phe Ala Gln Tyr Leu Ser Asp Lys Gln Val Pro Val Trp 165 170 175 Ile Arg His Val Leu Val Pro Gly Leu Thr Asp Phe Asp Glu His Leu 180 185 190 Val Lys Leu Gly Glu Phe Val Lys Thr Leu Lys Asn Val Asp Lys Phe 195 200 205

Glu Ile Leu Pro Tyr His Thr Met Gly Glu Phe Lys Trp Arg Glu Leu 210 215 220 Gly Ile Pro Tyr Pro Leu Glu Gly Val Lys Pro Pro Thr Ala Asp Arg 225 230 235 240 Val Lys Asn Ala Lys Ala Leu Met His Thr Glu Thr Tyr Gln Glu Tyr 245 250 255 Lys Asn Arg Ile Gly Val Lys Thr Leu Asp 260 265 <210> SEQ ID NO 85 <211> LENGTH: 1716 <212> TYPE: DNA <213> ORGANISM: Bacillus subtilis <400> SEQUENCE: 85 atgttgacaa aagcaacaaa agaacaaaaa tcccttgtga aaaacagagg ggcggagctt 60 gttgttgatt gcttagtgga gcaaggtgtc acacatgtat ttggcattcc aggtgcaaaa 120 attgatgcgg tatttgacgc tttacaagat aaaggacctg aaattatcgt tgcccggcac 180 gaacaaaacg cagcattcat ggcccaagca gtcggccgtt taactggaaa accgggagtc 240 gtgttagtca catcaggacc gggtgcctct aacttggcaa caggcctgct gacagcgaac 300 actgaaggag accctgtcgt tgcgcttgct ggaaacgtga tccgtgcaga tcgtttaaaa 360 cggacacatc aatctttgga taatgcggcg ctattccagc cgattacaaa atacagtgta 420 gaagttcaag atgtaaaaaa tataccggaa gctgttacaa atgcatttag gatagcgtca 480 gcagggcagg ctggggccgc ttttgtgagc tttccgcaag atgttgtgaa tgaagtcaca 540 aatacgaaaa acgtgcgtgc tgttgcagcg ccaaaactcg gtcctgcagc agatgatgca 600 atcagtgcgg ccatagcaaa aatccaaaca gcaaaacttc ctgtcgtttt ggtcggcatg 660 aaaggcggaa gaccggaagc aattaaagcg gttcgcaagc ttttgaaaaa ggttcagctt 720 ccatttgttg aaacatatca agctgccggt accctttcta gagatttaga ggatcaatat 780 tttggccgta tcggtttgtt ccgcaaccag cctggcgatt tactgctaga gcaggcagat 840 gttgttctga cgatcggcta tgacccgatt gaatatgatc cgaaattctg gaatatcaat 900 ggagaccgga caattatcca tttagacgag attatcgctg acattgatca tgcttaccag 960 cctgatcttg aattgatcgg tgacattccg tccacgatca atcatatcga acacgatgct 1020 gtgaaagtgg aatttgcaga gcgtgagcag aaaatccttt ctgatttaaa acaatatatg 1080 catgaaggtg agcaggtgcc tgcagattgg aaatcagaca gagcgcaccc tcttgaaatc 1140 gttaaagagt tgcgtaatgc agtcgatgat catgttacag taacttgcga tatcggttcg 1200 cacgccattt ggatgtcacg ttatttccgc agctacgagc cgttaacatt aatgatcagt 1260 aacggtatgc aaacactcgg cgttgcgctt ccttgggcaa tcggcgcttc attggtgaaa 1320 ccgggagaaa aagtggtttc tgtctctggt gacggcggtt tcttattctc agcaatggaa 1380 ttagagacag cagttcgact aaaagcacca attgtacaca ttgtatggaa cgacagcaca 1440 tatgacatgg ttgcattcca gcaattgaaa aaatataacc gtacatctgc ggtcgatttc 1500 ggaaatatcg atatcgtgaa atatgcggaa agcttcggag caactggctt gcgcgtagaa 1560 tcaccagacc agctggcaga tgttctgcgt caaggcatga acgctgaagg tcctgtcatc 1620 atcgatgtcc cggttgacta cagtgataac attaatttag caagtgacaa gcttccgaaa 1680 gaattcgggg aactcatgaa aacgaaagct ctctag 1716 <210> SEQ ID NO 86 <211> LENGTH: 571 <212> TYPE: PRT <213> ORGANISM: Bacillus subtilis <400> SEQUENCE: 86 Met Leu Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg 1 5 10 15 Gly Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His 20 25 30 Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu 35 40 45 Gln Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala 50 55 60 Ala Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val 65 70 75 80 Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu 85 90 95 Leu Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn 100 105 110 Val Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn 115 120 125 Ala Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp 130 135 140 Val Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser 145 150 155 160 Ala Gly Gln Ala Gly Ala Ala Phe Val Ser Phe Pro Gln Asp Val Val 165 170 175 Asn Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys 180 185 190 Leu Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ile Ala Lys Ile 195 200 205 Gln Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg 210 215 220 Pro Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu 225 230 235 240 Pro Phe Val Glu Thr Tyr Gln Ala Ala Gly Thr Leu Ser Arg Asp Leu 245 250 255 Glu Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly 260 265 270 Asp Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp 275 280 285 Pro Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr 290 295 300 Ile Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln 305 310 315 320 Pro Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile 325 330 335 Glu His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile 340 345 350 Leu Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala 355 360 365 Asp Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu 370 375 380 Arg Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser 385 390 395 400 His Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr 405 410 415 Leu Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp 420 425 430 Ala Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Val 435 440 445 Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala 450 455 460 Val Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr 465 470 475 480 Tyr Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser 485 490 495 Ala Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe 500 505 510 Gly Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val 515 520 525 Leu Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro 530 535 540 Val Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys 545 550 555 560 Glu Phe Gly Glu Leu Met Lys Thr Lys Ala Leu 565 570 <210> SEQ ID NO 87 <211> LENGTH: 1716 <212> TYPE: DNA <213> ORGANISM: artificial sequence <220> FEATURE: <223> OTHER INFORMATION: codon optimized coding region for expression in Lactobacillus plantarum <400> SEQUENCE: 87 atgttgacca aggctaccaa agaacaaaag agtttagtca aaaaccgtgg tgctgaatta 60 gtcgtggatt gtttggttga acaaggtgtg acgcatgttt ttggtattcc aggagctaaa 120 attgatgccg tttttgatgc gttacaagat aagggtccag aaattattgt ggcacgtcat 180 gaacaaaatg cagcgtttat ggctcaagca gttggtcggt tgactggcaa accaggtgtg 240 gttttagtga cgtcaggtcc aggtgcgagt aatttagcga ctggcttgtt aacggcgaat 300 actgaaggtg atccagtcgt tgctttggca ggcaatgtca ttcgtgccga tcgtttaaag 360 cggacccatc agagtttgga taatgcagcc ttgtttcaac cgattacgaa atattcagtt 420 gaagtccaag atgtcaagaa tattccagaa gcggttacga atgcgtttcg tattgcatca 480 gctggccaag caggcgcagc gtttgtgagt tttccacaag atgtcgtgaa tgaagttact 540 aacaccaaga atgtccgtgc agtcgcagct ccaaagttag gtccagcagc tgacgatgcc 600 attagtgcag ctattgccaa aattcagact gcaaaattgc cggttgtgtt agttggcatg 660 aaaggtggtc gtccagaagc cattaaagcg gttcgtaagt tattgaaaaa ggttcaatta 720 ccatttgttg aaacgtatca agctgcaggt acgttaagtc gtgacttaga agatcaatat 780 tttggtcgga ttggtttgtt tcgtaatcaa ccaggtgatt tgttattaga acaagctgat 840 gtggttttaa ctattggcta tgatccgatt gaatatgatc caaagttttg gaatattaat 900 ggtgatcgta ccatcattca tttggatgaa atcattgctg atattgatca cgcttatcaa 960 ccggatttgg aattaattgg tgacattcca agtacgatta atcacattga acatgatgct 1020 gtgaaggttg agtttgcgga acgggaacag aaaattttat cagatttgaa gcaatatatg 1080 catgaaggtg aacaagtgcc agcagattgg aagtcagatc gggcccatcc attagaaatt 1140 gttaaagaat tacggaatgc agtggacgat catgtgaccg tgacttgtga tattggtagt 1200 catgctattt ggatgagtcg ttactttcgg tcatatgaac cgttaacttt aatgatttca 1260 aacggtatgc aaactttagg tgttgccttg ccatgggcca ttggtgcgtc attggtcaaa 1320 ccaggtgaaa aggtcgtgtc agtcagtgga gatggtggct tcttattcag tgctatggaa 1380

ttagaaaccg ctgtgcggtt gaaggcaccg attgtgcata ttgtgtggaa cgatagtact 1440 tatgatatgg tcgcatttca acagttgaag aaatataatc gtacctcagc agtggatttt 1500 ggtaatatcg atattgtcaa gtatgccgaa agttttggtg ccaccggttt gcgtgtcgaa 1560 tcaccagatc aattagctga tgtcttgcgt caaggtatga atgcggaagg cccagttatt 1620 attgatgtgc cagttgatta cagtgataac attaatttag ctagtgataa gttgccgaaa 1680 gaatttggtg aattaatgaa gacgaaagcg ttataa 1716 <210> SEQ ID NO 88 <211> LENGTH: 1680 <212> TYPE: DNA <213> ORGANISM: Klebsiella pneumoniae <400> SEQUENCE: 88 atggacaaac agtatccggt acgccagtgg gcgcacggcg ccgatctcgt cgtcagtcag 60 ctggaagctc agggagtacg ccaggtgttc ggcatccccg gcgccaaaat cgacaaggtc 120 tttgattcac tgctggattc ctccattcgc attattccgg tacgccacga agccaacgcc 180 gcatttatgg ccgccgccgt cggacgcatt accggcaaag cgggcgtggc gctggtcacc 240 tccggtccgg gctgttccaa cctgatcacc ggcatggcca ccgcgaacag cgaaggcgac 300 ccggtggtgg ccctgggcgg cgcggtaaaa cgcgccgata aagcgaagca ggtccaccag 360 agtatggata cggtggcgat gttcagcccg gtcaccaaat acgccatcga ggtgacggcg 420 ccggatgcgc tggcggaagt ggtctccaac gccttccgcg ccgccgagca gggccggccg 480 ggcagcgcgt tcgttagcct gccgcaggat gtggtcgatg gcccggtcag cggcaaagtg 540 ctgccggcca gcggggcccc gcagatgggc gccgcgccgg atgatgccat cgaccaggtg 600 gcgaagctta tcgcccaggc gaagaacccg atcttcctgc tcggcctgat ggccagccag 660 ccggaaaaca gcaaggcgct gcgccgtttg ctggagacca gccatattcc agtcaccagc 720 acctatcagg ccgccggagc ggtgaatcag gataacttct ctcgcttcgc cggccgggtt 780 gggctgttta acaaccaggc cggggaccgt ctgctgcagc tcgccgacct ggtgatctgc 840 atcggctaca gcccggtgga atacgaaccg gcgatgtgga acagcggcaa cgcgacgctg 900 gtgcacatcg acgtgctgcc cgcctatgaa gagcgcaact acaccccgga tgtcgagctg 960 gtgggcgata tcgccggcac tctcaacaag ctggcgcaaa atatcgatca tcggctggtg 1020 ctctccccgc aggcggcgga gatcctccgc gaccgccagc accagcgcga gctgctggac 1080 cgccgcggcg cgcagctcaa ccagtttgcc ctgcatcccc tgcgcatcgt tcgcgccatg 1140 caggatatcg tcaacagcga cgtcacgttg accgtggaca tgggcagctt ccatatctgg 1200 attgcccgct acctgtacac gttccgcgcc cgtcaggtga tgatctccaa cggccagcag 1260 accatgggcg tcgccctgcc ctgggctatc ggcgcctggc tggtcaatcc tgagcgcaaa 1320 gtggtctccg tctccggcga cggcggcttc ctgcagtcga gcatggagct ggagaccgcc 1380 gtccgcctga aagccaacgt gctgcatctt atctgggtcg ataacggcta caacatggtc 1440 gctatccagg aagagaaaaa atatcagcgc ctgtccggcg tcgagtttgg gccgatggat 1500 tttaaagcct atgccgaatc cttcggcgcg aaagggtttg ccgtggaaag cgccgaggcg 1560 ctggagccga ccctgcgcgc ggcgatggac gtcgacggcc cggcggtagt ggccatcccg 1620 gtggattatc gcgataaccc gctgctgatg ggccagctgc atctgagtca gattctgtaa 1680 <210> SEQ ID NO 89 <211> LENGTH: 559 <212> TYPE: PRT <213> ORGANISM: Klebsiella pneumoniae <400> SEQUENCE: 89 Met Asp Lys Gln Tyr Pro Val Arg Gln Trp Ala His Gly Ala Asp Leu 1 5 10 15 Val Val Ser Gln Leu Glu Ala Gln Gly Val Arg Gln Val Phe Gly Ile 20 25 30 Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Ser Leu Leu Asp Ser Ser 35 40 45 Ile Arg Ile Ile Pro Val Arg His Glu Ala Asn Ala Ala Phe Met Ala 50 55 60 Ala Ala Val Gly Arg Ile Thr Gly Lys Ala Gly Val Ala Leu Val Thr 65 70 75 80 Ser Gly Pro Gly Cys Ser Asn Leu Ile Thr Gly Met Ala Thr Ala Asn 85 90 95 Ser Glu Gly Asp Pro Val Val Ala Leu Gly Gly Ala Val Lys Arg Ala 100 105 110 Asp Lys Ala Lys Gln Val His Gln Ser Met Asp Thr Val Ala Met Phe 115 120 125 Ser Pro Val Thr Lys Tyr Ala Ile Glu Val Thr Ala Pro Asp Ala Leu 130 135 140 Ala Glu Val Val Ser Asn Ala Phe Arg Ala Ala Glu Gln Gly Arg Pro 145 150 155 160 Gly Ser Ala Phe Val Ser Leu Pro Gln Asp Val Val Asp Gly Pro Val 165 170 175 Ser Gly Lys Val Leu Pro Ala Ser Gly Ala Pro Gln Met Gly Ala Ala 180 185 190 Pro Asp Asp Ala Ile Asp Gln Val Ala Lys Leu Ile Ala Gln Ala Lys 195 200 205 Asn Pro Ile Phe Leu Leu Gly Leu Met Ala Ser Gln Pro Glu Asn Ser 210 215 220 Lys Ala Leu Arg Arg Leu Leu Glu Thr Ser His Ile Pro Val Thr Ser 225 230 235 240 Thr Tyr Gln Ala Ala Gly Ala Val Asn Gln Asp Asn Phe Ser Arg Phe 245 250 255 Ala Gly Arg Val Gly Leu Phe Asn Asn Gln Ala Gly Asp Arg Leu Leu 260 265 270 Gln Leu Ala Asp Leu Val Ile Cys Ile Gly Tyr Ser Pro Val Glu Tyr 275 280 285 Glu Pro Ala Met Trp Asn Ser Gly Asn Ala Thr Leu Val His Ile Asp 290 295 300 Val Leu Pro Ala Tyr Glu Glu Arg Asn Tyr Thr Pro Asp Val Glu Leu 305 310 315 320 Val Gly Asp Ile Ala Gly Thr Leu Asn Lys Leu Ala Gln Asn Ile Asp 325 330 335 His Arg Leu Val Leu Ser Pro Gln Ala Ala Glu Ile Leu Arg Asp Arg 340 345 350 Gln His Gln Arg Glu Leu Leu Asp Arg Arg Gly Ala Gln Leu Asn Gln 355 360 365 Phe Ala Leu His Pro Leu Arg Ile Val Arg Ala Met Gln Asp Ile Val 370 375 380 Asn Ser Asp Val Thr Leu Thr Val Asp Met Gly Ser Phe His Ile Trp 385 390 395 400 Ile Ala Arg Tyr Leu Tyr Thr Phe Arg Ala Arg Gln Val Met Ile Ser 405 410 415 Asn Gly Gln Gln Thr Met Gly Val Ala Leu Pro Trp Ala Ile Gly Ala 420 425 430 Trp Leu Val Asn Pro Glu Arg Lys Val Val Ser Val Ser Gly Asp Gly 435 440 445 Gly Phe Leu Gln Ser Ser Met Glu Leu Glu Thr Ala Val Arg Leu Lys 450 455 460 Ala Asn Val Leu His Leu Ile Trp Val Asp Asn Gly Tyr Asn Met Val 465 470 475 480 Ala Ile Gln Glu Glu Lys Lys Tyr Gln Arg Leu Ser Gly Val Glu Phe 485 490 495 Gly Pro Met Asp Phe Lys Ala Tyr Ala Glu Ser Phe Gly Ala Lys Gly 500 505 510 Phe Ala Val Glu Ser Ala Glu Ala Leu Glu Pro Thr Leu Arg Ala Ala 515 520 525 Met Asp Val Asp Gly Pro Ala Val Val Ala Ile Pro Val Asp Tyr Arg 530 535 540 Asp Asn Pro Leu Leu Met Gly Gln Leu His Leu Ser Gln Ile Leu 545 550 555 <210> SEQ ID NO 90 <211> LENGTH: 1665 <212> TYPE: DNA <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 90 atgtctgaga aacaatttgg ggcgaacttg gttgtcgata gtttgattaa ccataaagtg 60 aagtatgtat ttgggattcc aggagcaaaa attgaccggg tttttgattt attagaaaat 120 gaagaaggcc ctcaaatggt cgtgactcgt catgagcaag gagctgcttt catggctcaa 180 gctgtcggtc gtttaactgg cgaacctggt gtagtagttg ttacgagtgg gcctggtgta 240 tcaaaccttg cgactccgct tttgaccgcg acatcagaag gtgatgctat tttggctatc 300 ggtggacaag ttaaacgaag tgaccgtctt aaacgtgcgc accaatcaat ggataatgct 360 ggaatgatgc aatcagcaac aaaatattca gcagaagttc ttgaccctaa tacactttct 420 gaatcaattg ccaacgctta tcgtattgca aaatcaggac atccaggtgc aactttctta 480 tcaatccccc aagatgtaac ggatgccgaa gtatcaatca aagccattca accactttca 540 gaccctaaaa tggggaatgc ctctattgat gacattaatt atttagcaca agcaattaaa 600 aatgctgtat tgccagtaat tttggttgga gctggtgctt cagatgctaa agtcgcttca 660 tccttgcgta atctattgac tcatgttaat attcctgtcg ttgaaacatt ccaaggtgca 720 ggggttattt cacatgattt agaacatact ttttatggac gtatcggtct tttccgcaat 780 caaccaggcg atatgcttct gaaacgttct gaccttgtta ttgctgttgg ttatgaccca 840 attgaatatg aagctcgtaa ctggaatgca gaaattgata gtcgaattat cgttattgat 900 aatgccattg ctgaaattga tacttactac caaccagagc gtgaattaat tggtgatatc 960 gcagcaacat tggataatct tttaccagct gttcgtggct acaaaattcc aaaaggaaca 1020 aaagattatc tcgatggcct tcatgaagtt gctgagcaac acgaatttga tactgaaaat 1080 actgaagaag gtagaatgca ccctcttgat ttggtcagca ctttccaaga aatcgtcaag 1140 gatgatgaaa cagtaaccgt tgacgtaggt tcactctaca tttggatggc acgtcatttc 1200 aaatcatacg aaccacgtca tctcctcttc tcaaacggaa tgcaaacact cggagttgca 1260 cttccttggg caattacagc cgcattgttg cgcccaggta aaaaagttta ttcacactct 1320 ggtgatggag gcttcctttt cacagggcaa gaattggaaa cagctgtacg tttgaatctt 1380 ccaatcgttc aaattatctg gaatgacggc cattatgata tggttaaatt ccaagaagaa 1440 atgaaatatg gtcgttcagc agccgttgat tttggctatg ttgattacgt aaaatatgct 1500 gaagcaatga gagcaaaagg ttaccgtgca cacagcaaag aagaacttgc tgaaattctc 1560 aaatcaatcc cagatactac tggaccggtg gtaattgacg ttcctttgga ctattctgat 1620

aacattaaat tagcagaaaa attattgcct gaagagtttt attga 1665 <210> SEQ ID NO 91 <211> LENGTH: 554 <212> TYPE: PRT <213> ORGANISM: Lactococcus lactis <400> SEQUENCE: 91 Met Ser Glu Lys Gln Phe Gly Ala Asn Leu Val Val Asp Ser Leu Ile 1 5 10 15 Asn His Lys Val Lys Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile Asp 20 25 30 Arg Val Phe Asp Leu Leu Glu Asn Glu Glu Gly Pro Gln Met Val Val 35 40 45 Thr Arg His Glu Gln Gly Ala Ala Phe Met Ala Gln Ala Val Gly Arg 50 55 60 Leu Thr Gly Glu Pro Gly Val Val Val Val Thr Ser Gly Pro Gly Val 65 70 75 80 Ser Asn Leu Ala Thr Pro Leu Leu Thr Ala Thr Ser Glu Gly Asp Ala 85 90 95 Ile Leu Ala Ile Gly Gly Gln Val Lys Arg Ser Asp Arg Leu Lys Arg 100 105 110 Ala His Gln Ser Met Asp Asn Ala Gly Met Met Gln Ser Ala Thr Lys 115 120 125 Tyr Ser Ala Glu Val Leu Asp Pro Asn Thr Leu Ser Glu Ser Ile Ala 130 135 140 Asn Ala Tyr Arg Ile Ala Lys Ser Gly His Pro Gly Ala Thr Phe Leu 145 150 155 160 Ser Ile Pro Gln Asp Val Thr Asp Ala Glu Val Ser Ile Lys Ala Ile 165 170 175 Gln Pro Leu Ser Asp Pro Lys Met Gly Asn Ala Ser Ile Asp Asp Ile 180 185 190 Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val Ile Leu 195 200 205 Val Gly Ala Gly Ala Ser Asp Ala Lys Val Ala Ser Ser Leu Arg Asn 210 215 220 Leu Leu Thr His Val Asn Ile Pro Val Val Glu Thr Phe Gln Gly Ala 225 230 235 240 Gly Val Ile Ser His Asp Leu Glu His Thr Phe Tyr Gly Arg Ile Gly 245 250 255 Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg Ser Asp Leu 260 265 270 Val Ile Ala Val Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Arg Asn Trp 275 280 285 Asn Ala Glu Ile Asp Ser Arg Ile Ile Val Ile Asp Asn Ala Ile Ala 290 295 300 Glu Ile Asp Thr Tyr Tyr Gln Pro Glu Arg Glu Leu Ile Gly Asp Ile 305 310 315 320 Ala Ala Thr Leu Asp Asn Leu Leu Pro Ala Val Arg Gly Tyr Lys Ile 325 330 335 Pro Lys Gly Thr Lys Asp Tyr Leu Asp Gly Leu His Glu Val Ala Glu 340 345 350 Gln His Glu Phe Asp Thr Glu Asn Thr Glu Glu Gly Arg Met His Pro 355 360 365 Leu Asp Leu Val Ser Thr Phe Gln Glu Ile Val Lys Asp Asp Glu Thr 370 375 380 Val Thr Val Asp Val Gly Ser Leu Tyr Ile Trp Met Ala Arg His Phe 385 390 395 400 Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr 405 410 415 Leu Gly Val Ala Leu Pro Trp Ala Ile Thr Ala Ala Leu Leu Arg Pro 420 425 430 Gly Lys Lys Val Tyr Ser His Ser Gly Asp Gly Gly Phe Leu Phe Thr 435 440 445 Gly Gln Glu Leu Glu Thr Ala Val Arg Leu Asn Leu Pro Ile Val Gln 450 455 460 Ile Ile Trp Asn Asp Gly His Tyr Asp Met Val Lys Phe Gln Glu Glu 465 470 475 480 Met Lys Tyr Gly Arg Ser Ala Ala Val Asp Phe Gly Tyr Val Asp Tyr 485 490 495 Val Lys Tyr Ala Glu Ala Met Arg Ala Lys Gly Tyr Arg Ala His Ser 500 505 510 Lys Glu Glu Leu Ala Glu Ile Leu Lys Ser Ile Pro Asp Thr Thr Gly 515 520 525 Pro Val Val Ile Asp Val Pro Leu Asp Tyr Ser Asp Asn Ile Lys Leu 530 535 540 Ala Glu Lys Leu Leu Pro Glu Glu Phe Tyr 545 550 <210> SEQ ID NO 92 <211> LENGTH: 1665 <212> TYPE: DNA <213> ORGANISM: Staphylococcus aureus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1665) <400> SEQUENCE: 92 atg act gat aaa aag tac act gca gcc gat atg gtt att gat act ttg 48 Met Thr Asp Lys Lys Tyr Thr Ala Ala Asp Met Val Ile Asp Thr Leu 1 5 10 15 aaa aat aat ggg gta gaa tat gtt ttt ggt att ccg ggt gca aag ata 96 Lys Asn Asn Gly Val Glu Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile 20 25 30 gac tat cta ttt aat gct tta att gat gat ggt cct gaa ctt att gtc 144 Asp Tyr Leu Phe Asn Ala Leu Ile Asp Asp Gly Pro Glu Leu Ile Val 35 40 45 act cgt cat gaa caa aat gct gca atg atg gca caa ggt att gga aga 192 Thr Arg His Glu Gln Asn Ala Ala Met Met Ala Gln Gly Ile Gly Arg 50 55 60 tta aca ggt aaa ccg ggt gta gta ctt gtt aca agt ggc cct ggt gta 240 Leu Thr Gly Lys Pro Gly Val Val Leu Val Thr Ser Gly Pro Gly Val 65 70 75 80 agt aat tta acg act gga cta tta aca gct aca tct gaa ggg gat cct 288 Ser Asn Leu Thr Thr Gly Leu Leu Thr Ala Thr Ser Glu Gly Asp Pro 85 90 95 gta tta gcg tta ggt ggc caa gtg aaa cgt aat gat tta tta cga tta 336 Val Leu Ala Leu Gly Gly Gln Val Lys Arg Asn Asp Leu Leu Arg Leu 100 105 110 acg cat caa agt att gat aat gct gcg cta tta aaa tat tca tca aaa 384 Thr His Gln Ser Ile Asp Asn Ala Ala Leu Leu Lys Tyr Ser Ser Lys 115 120 125 tac agt gaa gaa gta caa gat cct gaa tca tta tca gaa gtt atg aca 432 Tyr Ser Glu Glu Val Gln Asp Pro Glu Ser Leu Ser Glu Val Met Thr 130 135 140 aat gca att cga att gct act tca gga aaa aat ggc gca agt ttt att 480 Asn Ala Ile Arg Ile Ala Thr Ser Gly Lys Asn Gly Ala Ser Phe Ile 145 150 155 160 agt att ccg caa gac gtt att tct tca cca gtt gaa tct aaa gct ata 528 Ser Ile Pro Gln Asp Val Ile Ser Ser Pro Val Glu Ser Lys Ala Ile 165 170 175 tca ctt tgc caa aaa cca aat tta gga gta ccg agt gaa caa gat att 576 Ser Leu Cys Gln Lys Pro Asn Leu Gly Val Pro Ser Glu Gln Asp Ile 180 185 190 aat gat gtc att gaa gcg att aaa aat gca tca ttt cct gtt tta tta 624 Asn Asp Val Ile Glu Ala Ile Lys Asn Ala Ser Phe Pro Val Leu Leu 195 200 205 gct ggt atg aga agt tca agt gca gaa gaa aca aat gcc att cgc aaa 672 Ala Gly Met Arg Ser Ser Ser Ala Glu Glu Thr Asn Ala Ile Arg Lys 210 215 220 tta gtt gag cgc acg aat tta cca gtt gta gaa aca ttc caa ggt gca 720 Leu Val Glu Arg Thr Asn Leu Pro Val Val Glu Thr Phe Gln Gly Ala 225 230 235 240 ggt gta att agt cgt gaa tta gaa aat cat ttc ttc ggt cgt gtg ggc 768 Gly Val Ile Ser Arg Glu Leu Glu Asn His Phe Phe Gly Arg Val Gly 245 250 255 tta ttc cgc aat caa gtt ggt gat gaa tta tta cgt aaa agt gat tta 816 Leu Phe Arg Asn Gln Val Gly Asp Glu Leu Leu Arg Lys Ser Asp Leu 260 265 270 gtt gtt aca atc ggt tat gat cca att gaa tac gaa gct agt aac tgg 864 Val Val Thr Ile Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Ser Asn Trp 275 280 285 aat aaa gaa tta gaa aca caa att atc aat att gac gaa gtt caa gct 912 Asn Lys Glu Leu Glu Thr Gln Ile Ile Asn Ile Asp Glu Val Gln Ala 290 295 300 gaa att act aat tat atg caa ccg aaa aaa gag ttg att ggt aat att 960 Glu Ile Thr Asn Tyr Met Gln Pro Lys Lys Glu Leu Ile Gly Asn Ile 305 310 315 320 gct aaa acg att gaa atg att tct gaa aaa gtg gat gag cca ttt ata 1008 Ala Lys Thr Ile Glu Met Ile Ser Glu Lys Val Asp Glu Pro Phe Ile 325 330 335 aat caa caa cat tta gac gaa tta gaa caa tta aga aca cat att gat 1056 Asn Gln Gln His Leu Asp Glu Leu Glu Gln Leu Arg Thr His Ile Asp 340 345 350 gaa gaa act ggt att aaa gcg acg cat gaa gaa gga att cta cat cca 1104 Glu Glu Thr Gly Ile Lys Ala Thr His Glu Glu Gly Ile Leu His Pro 355 360 365 gtg gaa att att gaa tct atg caa aag gta tta act gat gat act act 1152 Val Glu Ile Ile Glu Ser Met Gln Lys Val Leu Thr Asp Asp Thr Thr 370 375 380 gta aca gtt gat gtt gga agt cac tat att tgg atg gca cgt aat ttc 1200 Val Thr Val Asp Val Gly Ser His Tyr Ile Trp Met Ala Arg Asn Phe 385 390 395 400 aga agt tac aat cca aga cat tta tta ttt agc aat ggt atg caa acg 1248 Arg Ser Tyr Asn Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr 405 410 415 ctt ggt gta gca tta ccg tgg gca att tca gct gca ctt gtg cgc cct 1296 Leu Gly Val Ala Leu Pro Trp Ala Ile Ser Ala Ala Leu Val Arg Pro 420 425 430 aat acg caa gtt gtg tcc gtt gct ggc gat ggt ggc ttt tta ttt tca 1344 Asn Thr Gln Val Val Ser Val Ala Gly Asp Gly Gly Phe Leu Phe Ser 435 440 445 tca caa gat tta gaa acg gcc gta cgt aaa aat tta aat atc atc cag 1392 Ser Gln Asp Leu Glu Thr Ala Val Arg Lys Asn Leu Asn Ile Ile Gln 450 455 460 ctt att tgg aat gat gga aaa tat aac atg gtt gaa ttc caa gaa gaa 1440 Leu Ile Trp Asn Asp Gly Lys Tyr Asn Met Val Glu Phe Gln Glu Glu 465 470 475 480 atg aaa tat aaa cgt tcg tca ggt gta gac ttc ggt cct gta gat ttt 1488 Met Lys Tyr Lys Arg Ser Ser Gly Val Asp Phe Gly Pro Val Asp Phe 485 490 495

gta aaa tat gca gaa tca ttt ggc gcg aaa ggt tta cga gtt act aat 1536 Val Lys Tyr Ala Glu Ser Phe Gly Ala Lys Gly Leu Arg Val Thr Asn 500 505 510 caa gaa gaa tta gaa gcg gca att aaa gag ggc tat gaa aca gat ggt 1584 Gln Glu Glu Leu Glu Ala Ala Ile Lys Glu Gly Tyr Glu Thr Asp Gly 515 520 525 cca gta tta att gat ata cct gta aat tac aaa gat aat atc aaa ctt 1632 Pro Val Leu Ile Asp Ile Pro Val Asn Tyr Lys Asp Asn Ile Lys Leu 530 535 540 tca aca aat atg tta cct gac gta ttt aac taa 1665 Ser Thr Asn Met Leu Pro Asp Val Phe Asn 545 550 <210> SEQ ID NO 93 <211> LENGTH: 554 <212> TYPE: PRT <213> ORGANISM: Staphylococcus aureus <400> SEQUENCE: 93 Met Thr Asp Lys Lys Tyr Thr Ala Ala Asp Met Val Ile Asp Thr Leu 1 5 10 15 Lys Asn Asn Gly Val Glu Tyr Val Phe Gly Ile Pro Gly Ala Lys Ile 20 25 30 Asp Tyr Leu Phe Asn Ala Leu Ile Asp Asp Gly Pro Glu Leu Ile Val 35 40 45 Thr Arg His Glu Gln Asn Ala Ala Met Met Ala Gln Gly Ile Gly Arg 50 55 60 Leu Thr Gly Lys Pro Gly Val Val Leu Val Thr Ser Gly Pro Gly Val 65 70 75 80 Ser Asn Leu Thr Thr Gly Leu Leu Thr Ala Thr Ser Glu Gly Asp Pro 85 90 95 Val Leu Ala Leu Gly Gly Gln Val Lys Arg Asn Asp Leu Leu Arg Leu 100 105 110 Thr His Gln Ser Ile Asp Asn Ala Ala Leu Leu Lys Tyr Ser Ser Lys 115 120 125 Tyr Ser Glu Glu Val Gln Asp Pro Glu Ser Leu Ser Glu Val Met Thr 130 135 140 Asn Ala Ile Arg Ile Ala Thr Ser Gly Lys Asn Gly Ala Ser Phe Ile 145 150 155 160 Ser Ile Pro Gln Asp Val Ile Ser Ser Pro Val Glu Ser Lys Ala Ile 165 170 175 Ser Leu Cys Gln Lys Pro Asn Leu Gly Val Pro Ser Glu Gln Asp Ile 180 185 190 Asn Asp Val Ile Glu Ala Ile Lys Asn Ala Ser Phe Pro Val Leu Leu 195 200 205 Ala Gly Met Arg Ser Ser Ser Ala Glu Glu Thr Asn Ala Ile Arg Lys 210 215 220 Leu Val Glu Arg Thr Asn Leu Pro Val Val Glu Thr Phe Gln Gly Ala 225 230 235 240 Gly Val Ile Ser Arg Glu Leu Glu Asn His Phe Phe Gly Arg Val Gly 245 250 255 Leu Phe Arg Asn Gln Val Gly Asp Glu Leu Leu Arg Lys Ser Asp Leu 260 265 270 Val Val Thr Ile Gly Tyr Asp Pro Ile Glu Tyr Glu Ala Ser Asn Trp 275 280 285 Asn Lys Glu Leu Glu Thr Gln Ile Ile Asn Ile Asp Glu Val Gln Ala 290 295 300 Glu Ile Thr Asn Tyr Met Gln Pro Lys Lys Glu Leu Ile Gly Asn Ile 305 310 315 320 Ala Lys Thr Ile Glu Met Ile Ser Glu Lys Val Asp Glu Pro Phe Ile 325 330 335 Asn Gln Gln His Leu Asp Glu Leu Glu Gln Leu Arg Thr His Ile Asp 340 345 350 Glu Glu Thr Gly Ile Lys Ala Thr His Glu Glu Gly Ile Leu His Pro 355 360 365 Val Glu Ile Ile Glu Ser Met Gln Lys Val Leu Thr Asp Asp Thr Thr 370 375 380 Val Thr Val Asp Val Gly Ser His Tyr Ile Trp Met Ala Arg Asn Phe 385 390 395 400 Arg Ser Tyr Asn Pro Arg His Leu Leu Phe Ser Asn Gly Met Gln Thr 405 410 415 Leu Gly Val Ala Leu Pro Trp Ala Ile Ser Ala Ala Leu Val Arg Pro 420 425 430 Asn Thr Gln Val Val Ser Val Ala Gly Asp Gly Gly Phe Leu Phe Ser 435 440 445 Ser Gln Asp Leu Glu Thr Ala Val Arg Lys Asn Leu Asn Ile Ile Gln 450 455 460 Leu Ile Trp Asn Asp Gly Lys Tyr Asn Met Val Glu Phe Gln Glu Glu 465 470 475 480 Met Lys Tyr Lys Arg Ser Ser Gly Val Asp Phe Gly Pro Val Asp Phe 485 490 495 Val Lys Tyr Ala Glu Ser Phe Gly Ala Lys Gly Leu Arg Val Thr Asn 500 505 510 Gln Glu Glu Leu Glu Ala Ala Ile Lys Glu Gly Tyr Glu Thr Asp Gly 515 520 525 Pro Val Leu Ile Asp Ile Pro Val Asn Tyr Lys Asp Asn Ile Lys Leu 530 535 540 Ser Thr Asn Met Leu Pro Asp Val Phe Asn 545 550 <210> SEQ ID NO 94 <211> LENGTH: 1698 <212> TYPE: DNA <213> ORGANISM: Listeria monocytogenes <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1698) <400> SEQUENCE: 94 atg gcg aaa cta gaa aaa gac caa gaa aaa gta ata aca caa ggg aaa 48 Met Ala Lys Leu Glu Lys Asp Gln Glu Lys Val Ile Thr Gln Gly Lys 1 5 10 15 tca gga gcg gat tta gtt gta gac agc tta att aat caa ggt gtt acg 96 Ser Gly Ala Asp Leu Val Val Asp Ser Leu Ile Asn Gln Gly Val Thr 20 25 30 cat gta ttc ggg att ccg gga gcg aaa att gat aaa gtt ttt gat gtg 144 His Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Val 35 40 45 atg gaa gaa cgt gga cca gaa tta att gtc agt cgt cat gaa caa aat 192 Met Glu Glu Arg Gly Pro Glu Leu Ile Val Ser Arg His Glu Gln Asn 50 55 60 gcg gcg ttt atg gct gct gct atc ggt cgt cta acc ggg aaa cct ggt 240 Ala Ala Phe Met Ala Ala Ala Ile Gly Arg Leu Thr Gly Lys Pro Gly 65 70 75 80 gtt gta ctt gta act agt gga cct ggc gca tcg aat ctt gca aca ggg 288 Val Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly 85 90 95 ctt gta acc gca act gca gaa gga gat cca gtc gtt gcg att gct ggt 336 Leu Val Thr Ala Thr Ala Glu Gly Asp Pro Val Val Ala Ile Ala Gly 100 105 110 aac gta aca agg caa gac cgc tta aaa aga acc cac caa tca atg gat 384 Asn Val Thr Arg Gln Asp Arg Leu Lys Arg Thr His Gln Ser Met Asp 115 120 125 aat gca gca ctt ttc cgt ccg att aca aaa tac agc gaa gaa gta gtt 432 Asn Ala Ala Leu Phe Arg Pro Ile Thr Lys Tyr Ser Glu Glu Val Val 130 135 140 cac gcc gaa agt att cca gaa gca atc act aac gct ttt cgc tcg gca 480 His Ala Glu Ser Ile Pro Glu Ala Ile Thr Asn Ala Phe Arg Ser Ala 145 150 155 160 aca gaa cca aac caa ggc gct gct ttt gtc agt ttg cca caa gat atc 528 Thr Glu Pro Asn Gln Gly Ala Ala Phe Val Ser Leu Pro Gln Asp Ile 165 170 175 gtg aac gaa cca aac gta cca gta aaa gcg att cgc cca ctt gct aaa 576 Val Asn Glu Pro Asn Val Pro Val Lys Ala Ile Arg Pro Leu Ala Lys 180 185 190 cca gaa aat ggt cct gct tcc aaa gaa caa gtt gca aaa ctt gtt aca 624 Pro Glu Asn Gly Pro Ala Ser Lys Glu Gln Val Ala Lys Leu Val Thr 195 200 205 cgt ttg aaa aaa gcg aaa tta ccg gta ttg cta ttg ggt atg cga gca 672 Arg Leu Lys Lys Ala Lys Leu Pro Val Leu Leu Leu Gly Met Arg Ala 210 215 220 tct agt cca gaa gta act ggt gca att cgt cgc tta ctc caa aaa aca 720 Ser Ser Pro Glu Val Thr Gly Ala Ile Arg Arg Leu Leu Gln Lys Thr 225 230 235 240 agt atc cca gta gta gaa act ttc caa gca gct ggc gtc att tca cgc 768 Ser Ile Pro Val Val Glu Thr Phe Gln Ala Ala Gly Val Ile Ser Arg 245 250 255 gac tta gaa gat aac ttc ttt gga cgt gtt ggt ctg ttc cgc aac caa 816 Asp Leu Glu Asp Asn Phe Phe Gly Arg Val Gly Leu Phe Arg Asn Gln 260 265 270 cca ggg gat att ttg tta aat aaa gct gat tta gtt att aca gtg ggt 864 Pro Gly Asp Ile Leu Leu Asn Lys Ala Asp Leu Val Ile Thr Val Gly 275 280 285 tat gat cca att gaa tac gat cca aaa gct tgg aat gcc tct ggt gat 912 Tyr Asp Pro Ile Glu Tyr Asp Pro Lys Ala Trp Asn Ala Ser Gly Asp 290 295 300 aga acg att gtc cat tta gac gac att cgc gct gat att gat cat tat 960 Arg Thr Ile Val His Leu Asp Asp Ile Arg Ala Asp Ile Asp His Tyr 305 310 315 320 tac caa cca gtg aca gag cta gtc gga aac atc gcg ctt act tta gac 1008 Tyr Gln Pro Val Thr Glu Leu Val Gly Asn Ile Ala Leu Thr Leu Asp 325 330 335 cga gtg aat gcg aaa ttc agc ggt tta gaa tta gcg gaa aaa gaa ctt 1056 Arg Val Asn Ala Lys Phe Ser Gly Leu Glu Leu Ala Glu Lys Glu Leu 340 345 350 gaa aca tta aaa gaa ctt cat gct caa tta gaa gag cga gat gtt ccg 1104 Glu Thr Leu Lys Glu Leu His Ala Gln Leu Glu Glu Arg Asp Val Pro 355 360 365 cca gaa agt gat gaa act aac cga gta cat cca ttg tcg gtc att caa 1152 Pro Glu Ser Asp Glu Thr Asn Arg Val His Pro Leu Ser Val Ile Gln 370 375 380 aca cta cgt tcg gca att gat gac aac gta act gtg aca gtc gac gtt 1200 Thr Leu Arg Ser Ala Ile Asp Asp Asn Val Thr Val Thr Val Asp Val 385 390 395 400 ggt tca cat tat att tgg atg gca cgt cat ttc cgc tcc tat gaa cca 1248 Gly Ser His Tyr Ile Trp Met Ala Arg His Phe Arg Ser Tyr Glu Pro 405 410 415 cgc cgt ctg ctt ttc agt aac ggt atg caa acg ctt ggt gtt gcg ctt 1296 Arg Arg Leu Leu Phe Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu 420 425 430 cct tgg gga att gct gca aca ctt gta cat ccg ggt gaa aaa gtg gtt 1344 Pro Trp Gly Ile Ala Ala Thr Leu Val His Pro Gly Glu Lys Val Val 435 440 445

tcg att tct ggt gac ggt ggt ttc tta ttt tcc gcg atg gaa tta gaa 1392 Ser Ile Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu 450 455 460 aca gct gtc cgc ttg cgt gcg cca ctt gta cac cta gta tgg aat gac 1440 Thr Ala Val Arg Leu Arg Ala Pro Leu Val His Leu Val Trp Asn Asp 465 470 475 480 gga agc tat gac atg gtt gct ttc caa caa aaa atg aaa tac ggc aaa 1488 Gly Ser Tyr Asp Met Val Ala Phe Gln Gln Lys Met Lys Tyr Gly Lys 485 490 495 gaa gca gct gtt cgt ttt ggc gat gtt gat atc gta aaa ttt gca gaa 1536 Glu Ala Ala Val Arg Phe Gly Asp Val Asp Ile Val Lys Phe Ala Glu 500 505 510 agt ttc gga gca aaa ggt ctt cgc gta aca aat cca gca gaa ctt tct 1584 Ser Phe Gly Ala Lys Gly Leu Arg Val Thr Asn Pro Ala Glu Leu Ser 515 520 525 gat gtg tta aaa gaa gcg ctt gaa aca gaa gga ccc gtc gtt gta gat 1632 Asp Val Leu Lys Glu Ala Leu Glu Thr Glu Gly Pro Val Val Val Asp 530 535 540 att cca att gat tac cgt gat aac atc aaa ctt ggc gaa act tta cta 1680 Ile Pro Ile Asp Tyr Arg Asp Asn Ile Lys Leu Gly Glu Thr Leu Leu 545 550 555 560 cct gac caa ttt tat taa 1698 Pro Asp Gln Phe Tyr 565 <210> SEQ ID NO 95 <211> LENGTH: 565 <212> TYPE: PRT <213> ORGANISM: Listeria monocytogenes <400> SEQUENCE: 95 Met Ala Lys Leu Glu Lys Asp Gln Glu Lys Val Ile Thr Gln Gly Lys 1 5 10 15 Ser Gly Ala Asp Leu Val Val Asp Ser Leu Ile Asn Gln Gly Val Thr 20 25 30 His Val Phe Gly Ile Pro Gly Ala Lys Ile Asp Lys Val Phe Asp Val 35 40 45 Met Glu Glu Arg Gly Pro Glu Leu Ile Val Ser Arg His Glu Gln Asn 50 55 60 Ala Ala Phe Met Ala Ala Ala Ile Gly Arg Leu Thr Gly Lys Pro Gly 65 70 75 80 Val Val Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly 85 90 95 Leu Val Thr Ala Thr Ala Glu Gly Asp Pro Val Val Ala Ile Ala Gly 100 105 110 Asn Val Thr Arg Gln Asp Arg Leu Lys Arg Thr His Gln Ser Met Asp 115 120 125 Asn Ala Ala Leu Phe Arg Pro Ile Thr Lys Tyr Ser Glu Glu Val Val 130 135 140 His Ala Glu Ser Ile Pro Glu Ala Ile Thr Asn Ala Phe Arg Ser Ala 145 150 155 160 Thr Glu Pro Asn Gln Gly Ala Ala Phe Val Ser Leu Pro Gln Asp Ile 165 170 175 Val Asn Glu Pro Asn Val Pro Val Lys Ala Ile Arg Pro Leu Ala Lys 180 185 190 Pro Glu Asn Gly Pro Ala Ser Lys Glu Gln Val Ala Lys Leu Val Thr 195 200 205 Arg Leu Lys Lys Ala Lys Leu Pro Val Leu Leu Leu Gly Met Arg Ala 210 215 220 Ser Ser Pro Glu Val Thr Gly Ala Ile Arg Arg Leu Leu Gln Lys Thr 225 230 235 240 Ser Ile Pro Val Val Glu Thr Phe Gln Ala Ala Gly Val Ile Ser Arg 245 250 255 Asp Leu Glu Asp Asn Phe Phe Gly Arg Val Gly Leu Phe Arg Asn Gln 260 265 270 Pro Gly Asp Ile Leu Leu Asn Lys Ala Asp Leu Val Ile Thr Val Gly 275 280 285 Tyr Asp Pro Ile Glu Tyr Asp Pro Lys Ala Trp Asn Ala Ser Gly Asp 290 295 300 Arg Thr Ile Val His Leu Asp Asp Ile Arg Ala Asp Ile Asp His Tyr 305 310 315 320 Tyr Gln Pro Val Thr Glu Leu Val Gly Asn Ile Ala Leu Thr Leu Asp 325 330 335 Arg Val Asn Ala Lys Phe Ser Gly Leu Glu Leu Ala Glu Lys Glu Leu 340 345 350 Glu Thr Leu Lys Glu Leu His Ala Gln Leu Glu Glu Arg Asp Val Pro 355 360 365 Pro Glu Ser Asp Glu Thr Asn Arg Val His Pro Leu Ser Val Ile Gln 370 375 380 Thr Leu Arg Ser Ala Ile Asp Asp Asn Val Thr Val Thr Val Asp Val 385 390 395 400 Gly Ser His Tyr Ile Trp Met Ala Arg His Phe Arg Ser Tyr Glu Pro 405 410 415 Arg Arg Leu Leu Phe Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu 420 425 430 Pro Trp Gly Ile Ala Ala Thr Leu Val His Pro Gly Glu Lys Val Val 435 440 445 Ser Ile Ser Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu 450 455 460 Thr Ala Val Arg Leu Arg Ala Pro Leu Val His Leu Val Trp Asn Asp 465 470 475 480 Gly Ser Tyr Asp Met Val Ala Phe Gln Gln Lys Met Lys Tyr Gly Lys 485 490 495 Glu Ala Ala Val Arg Phe Gly Asp Val Asp Ile Val Lys Phe Ala Glu 500 505 510 Ser Phe Gly Ala Lys Gly Leu Arg Val Thr Asn Pro Ala Glu Leu Ser 515 520 525 Asp Val Leu Lys Glu Ala Leu Glu Thr Glu Gly Pro Val Val Val Asp 530 535 540 Ile Pro Ile Asp Tyr Arg Asp Asn Ile Lys Leu Gly Glu Thr Leu Leu 545 550 555 560 Pro Asp Gln Phe Tyr 565 <210> SEQ ID NO 96 <211> LENGTH: 1680 <212> TYPE: DNA <213> ORGANISM: Streptococcus mutans <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1680) <400> SEQUENCE: 96 atg acc gaa ata aat aag gaa ggc tat ggg gct gac ctg att gta gac 48 Met Thr Glu Ile Asn Lys Glu Gly Tyr Gly Ala Asp Leu Ile Val Asp 1 5 10 15 agc ctc att aat cat gat gtc aac tat gtt ttt gga atc cct ggt gca 96 Ser Leu Ile Asn His Asp Val Asn Tyr Val Phe Gly Ile Pro Gly Ala 20 25 30 aaa att gat cgt gtc ttt gat acc tta gaa gat aag ggg cca gaa ctt 144 Lys Ile Asp Arg Val Phe Asp Thr Leu Glu Asp Lys Gly Pro Glu Leu 35 40 45 att gta gca cgc cat gag caa aat gct gct ttt atg gct caa gga att 192 Ile Val Ala Arg His Glu Gln Asn Ala Ala Phe Met Ala Gln Gly Ile 50 55 60 ggc cgt att act ggt gag cct ggt gtt gtg att aca acc agc ggt ccc 240 Gly Arg Ile Thr Gly Glu Pro Gly Val Val Ile Thr Thr Ser Gly Pro 65 70 75 80 ggt gtt tcc aat ctg gtg act ggt ctt gtt act gcg aca gct gag gga 288 Gly Val Ser Asn Leu Val Thr Gly Leu Val Thr Ala Thr Ala Glu Gly 85 90 95 gat cct gtc ctt gct att ggt ggt cag gtt aaa cgt gct gat ttg ctc 336 Asp Pro Val Leu Ala Ile Gly Gly Gln Val Lys Arg Ala Asp Leu Leu 100 105 110 aaa cgg gct cac cag tca atg aat aat gtt gct atg ctc gat ccc att 384 Lys Arg Ala His Gln Ser Met Asn Asn Val Ala Met Leu Asp Pro Ile 115 120 125 acc aaa tat tca gca gaa att cag gat ccc gca aca ctt tca gaa aat 432 Thr Lys Tyr Ser Ala Glu Ile Gln Asp Pro Ala Thr Leu Ser Glu Asn 130 135 140 att gct aat gcc tat cgt ttg gct aaa gca gga aag ccg gga gct agt 480 Ile Ala Asn Ala Tyr Arg Leu Ala Lys Ala Gly Lys Pro Gly Ala Ser 145 150 155 160 ttc tta tct att cct caa gat ata act gat agt cct gtt act gtc aag 528 Phe Leu Ser Ile Pro Gln Asp Ile Thr Asp Ser Pro Val Thr Val Lys 165 170 175 gcg att aag ccc ttg aca gat cct aaa cta ggt tca gcg tca gtt gct 576 Ala Ile Lys Pro Leu Thr Asp Pro Lys Leu Gly Ser Ala Ser Val Ala 180 185 190 gat att aat tat ttg gca cag gcc ata aaa aat gcg gtc ctt cct gtc 624 Asp Ile Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val 195 200 205 tta ctt tta gga aat ggt gcg tca acg gct gca gtt aca gct tct att 672 Leu Leu Leu Gly Asn Gly Ala Ser Thr Ala Ala Val Thr Ala Ser Ile 210 215 220 cgc cgt ttg tta gga gct gtc aag ctg cca gtc gtt gaa act ttc caa 720 Arg Arg Leu Leu Gly Ala Val Lys Leu Pro Val Val Glu Thr Phe Gln 225 230 235 240 gga gct ggt att gtt tca aga gat tta gaa gag gac act ttt ttt ggt 768 Gly Ala Gly Ile Val Ser Arg Asp Leu Glu Glu Asp Thr Phe Phe Gly 245 250 255 cgt gtg ggg ctt ttt cgt aat cag ccc gga gat atg ttg ctg aag cgt 816 Arg Val Gly Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg 260 265 270 tct gac tta gtt atc gct att ggc tat gat cct att gaa tat gaa gcg 864 Ser Asp Leu Val Ile Ala Ile Gly Tyr Asp Pro Ile Glu Tyr Glu Ala 275 280 285 cgc aat tgg aat gct gaa att tcg gct cgc att atc gtt att gat gtt 912 Arg Asn Trp Asn Ala Glu Ile Ser Ala Arg Ile Ile Val Ile Asp Val 290 295 300 gct cca gct gaa att gat act tat ttc caa cct gaa cgt gaa tta att 960 Ala Pro Ala Glu Ile Asp Thr Tyr Phe Gln Pro Glu Arg Glu Leu Ile 305 310 315 320 ggt gat ata gct gaa aca ctt gat tta ctc cta cct gct att agt ggc 1008 Gly Asp Ile Ala Glu Thr Leu Asp Leu Leu Leu Pro Ala Ile Ser Gly 325 330 335 tac tca ctt cca aaa ggt tct ctt gac tat ctc aaa ggc ctt cgt gat 1056 Tyr Ser Leu Pro Lys Gly Ser Leu Asp Tyr Leu Lys Gly Leu Arg Asp 340 345 350 aat gta gta gaa gat gtc aaa ttt gat aag aca gtc aaa tcc ggt ctg 1104 Asn Val Val Glu Asp Val Lys Phe Asp Lys Thr Val Lys Ser Gly Leu 355 360 365

gtt cat ccg ctt gat gtg att gat gtc ctt caa aag caa acg act gat 1152 Val His Pro Leu Asp Val Ile Asp Val Leu Gln Lys Gln Thr Thr Asp 370 375 380 gat atg aca gta acg gtt gat gtt ggc agc cat tat att tgg atg gct 1200 Asp Met Thr Val Thr Val Asp Val Gly Ser His Tyr Ile Trp Met Ala 385 390 395 400 cgt tat ttt aaa agc tat gaa gca cgg cac tta ctt ttc tca aat ggt 1248 Arg Tyr Phe Lys Ser Tyr Glu Ala Arg His Leu Leu Phe Ser Asn Gly 405 410 415 atg caa acc tta ggt gtt gct ttg cct tgg gca att tcg gca gct ctt 1296 Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala Ile Ser Ala Ala Leu 420 425 430 gta cgg cca aat gag aag att att tct att tca ggt gat ggt ggt ttc 1344 Val Arg Pro Asn Glu Lys Ile Ile Ser Ile Ser Gly Asp Gly Gly Phe 435 440 445 ctc ttt tct ggc caa gaa ttg gaa aca gct gtt cgt tta cat tta cca 1392 Leu Phe Ser Gly Gln Glu Leu Glu Thr Ala Val Arg Leu His Leu Pro 450 455 460 att gtt cat atc att tgg aat gat ggt aaa tat aat atg gtt gaa ttc 1440 Ile Val His Ile Ile Trp Asn Asp Gly Lys Tyr Asn Met Val Glu Phe 465 470 475 480 caa gaa gaa atg aaa tac ggc cgt tca gca ggt gtt gat ttt ggt cct 1488 Gln Glu Glu Met Lys Tyr Gly Arg Ser Ala Gly Val Asp Phe Gly Pro 485 490 495 gtt gat ttt gtc aag tat gct gat agt ttc ggt gct aaa ggt tac cgt 1536 Val Asp Phe Val Lys Tyr Ala Asp Ser Phe Gly Ala Lys Gly Tyr Arg 500 505 510 gct gat agt aaa gaa aag ttt gat caa gtt ctt caa aca gca ctc aag 1584 Ala Asp Ser Lys Glu Lys Phe Asp Gln Val Leu Gln Thr Ala Leu Lys 515 520 525 gaa gct gca aat ggc cca gtt ctc att gat gtt cca atg gac tat aaa 1632 Glu Ala Ala Asn Gly Pro Val Leu Ile Asp Val Pro Met Asp Tyr Lys 530 535 540 gat aat gta aaa ttg ggt gaa act att ttg cct gat gaa ttc tac taa 1680 Asp Asn Val Lys Leu Gly Glu Thr Ile Leu Pro Asp Glu Phe Tyr 545 550 555 <210> SEQ ID NO 97 <211> LENGTH: 559 <212> TYPE: PRT <213> ORGANISM: Streptococcus mutans <400> SEQUENCE: 97 Met Thr Glu Ile Asn Lys Glu Gly Tyr Gly Ala Asp Leu Ile Val Asp 1 5 10 15 Ser Leu Ile Asn His Asp Val Asn Tyr Val Phe Gly Ile Pro Gly Ala 20 25 30 Lys Ile Asp Arg Val Phe Asp Thr Leu Glu Asp Lys Gly Pro Glu Leu 35 40 45 Ile Val Ala Arg His Glu Gln Asn Ala Ala Phe Met Ala Gln Gly Ile 50 55 60 Gly Arg Ile Thr Gly Glu Pro Gly Val Val Ile Thr Thr Ser Gly Pro 65 70 75 80 Gly Val Ser Asn Leu Val Thr Gly Leu Val Thr Ala Thr Ala Glu Gly 85 90 95 Asp Pro Val Leu Ala Ile Gly Gly Gln Val Lys Arg Ala Asp Leu Leu 100 105 110 Lys Arg Ala His Gln Ser Met Asn Asn Val Ala Met Leu Asp Pro Ile 115 120 125 Thr Lys Tyr Ser Ala Glu Ile Gln Asp Pro Ala Thr Leu Ser Glu Asn 130 135 140 Ile Ala Asn Ala Tyr Arg Leu Ala Lys Ala Gly Lys Pro Gly Ala Ser 145 150 155 160 Phe Leu Ser Ile Pro Gln Asp Ile Thr Asp Ser Pro Val Thr Val Lys 165 170 175 Ala Ile Lys Pro Leu Thr Asp Pro Lys Leu Gly Ser Ala Ser Val Ala 180 185 190 Asp Ile Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro Val 195 200 205 Leu Leu Leu Gly Asn Gly Ala Ser Thr Ala Ala Val Thr Ala Ser Ile 210 215 220 Arg Arg Leu Leu Gly Ala Val Lys Leu Pro Val Val Glu Thr Phe Gln 225 230 235 240 Gly Ala Gly Ile Val Ser Arg Asp Leu Glu Glu Asp Thr Phe Phe Gly 245 250 255 Arg Val Gly Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys Arg 260 265 270 Ser Asp Leu Val Ile Ala Ile Gly Tyr Asp Pro Ile Glu Tyr Glu Ala 275 280 285 Arg Asn Trp Asn Ala Glu Ile Ser Ala Arg Ile Ile Val Ile Asp Val 290 295 300 Ala Pro Ala Glu Ile Asp Thr Tyr Phe Gln Pro Glu Arg Glu Leu Ile 305 310 315 320 Gly Asp Ile Ala Glu Thr Leu Asp Leu Leu Leu Pro Ala Ile Ser Gly 325 330 335 Tyr Ser Leu Pro Lys Gly Ser Leu Asp Tyr Leu Lys Gly Leu Arg Asp 340 345 350 Asn Val Val Glu Asp Val Lys Phe Asp Lys Thr Val Lys Ser Gly Leu 355 360 365 Val His Pro Leu Asp Val Ile Asp Val Leu Gln Lys Gln Thr Thr Asp 370 375 380 Asp Met Thr Val Thr Val Asp Val Gly Ser His Tyr Ile Trp Met Ala 385 390 395 400 Arg Tyr Phe Lys Ser Tyr Glu Ala Arg His Leu Leu Phe Ser Asn Gly 405 410 415 Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala Ile Ser Ala Ala Leu 420 425 430 Val Arg Pro Asn Glu Lys Ile Ile Ser Ile Ser Gly Asp Gly Gly Phe 435 440 445 Leu Phe Ser Gly Gln Glu Leu Glu Thr Ala Val Arg Leu His Leu Pro 450 455 460 Ile Val His Ile Ile Trp Asn Asp Gly Lys Tyr Asn Met Val Glu Phe 465 470 475 480 Gln Glu Glu Met Lys Tyr Gly Arg Ser Ala Gly Val Asp Phe Gly Pro 485 490 495 Val Asp Phe Val Lys Tyr Ala Asp Ser Phe Gly Ala Lys Gly Tyr Arg 500 505 510 Ala Asp Ser Lys Glu Lys Phe Asp Gln Val Leu Gln Thr Ala Leu Lys 515 520 525 Glu Ala Ala Asn Gly Pro Val Leu Ile Asp Val Pro Met Asp Tyr Lys 530 535 540 Asp Asn Val Lys Leu Gly Glu Thr Ile Leu Pro Asp Glu Phe Tyr 545 550 555 <210> SEQ ID NO 98 <211> LENGTH: 1683 <212> TYPE: DNA <213> ORGANISM: Streptococcus thermophilus <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1683) <400> SEQUENCE: 98 gtg ttc atg tca gaa gaa aag caa ttg tat ggt gca gat tta gtg gtt 48 Val Phe Met Ser Glu Glu Lys Gln Leu Tyr Gly Ala Asp Leu Val Val 1 5 10 15 gat agt ttg atc aac cat gat gtt gag tat gtc ttt ggg att cca ggc 96 Asp Ser Leu Ile Asn His Asp Val Glu Tyr Val Phe Gly Ile Pro Gly 20 25 30 gca aaa atc gat agg gtt ttt gat acc ttg gaa gat aag gga cct gaa 144 Ala Lys Ile Asp Arg Val Phe Asp Thr Leu Glu Asp Lys Gly Pro Glu 35 40 45 ttg att gtt gcc cgt cat gag caa aat gct gct ttt atg gct caa ggt 192 Leu Ile Val Ala Arg His Glu Gln Asn Ala Ala Phe Met Ala Gln Gly 50 55 60 gtt gga cgt att act ggg aaa cca ggt gta gta ttg gta aca tct ggt 240 Val Gly Arg Ile Thr Gly Lys Pro Gly Val Val Leu Val Thr Ser Gly 65 70 75 80 cca ggt gtc tcc aat ttg gct act ggt ttg gta aca gcg acg gat gaa 288 Pro Gly Val Ser Asn Leu Ala Thr Gly Leu Val Thr Ala Thr Asp Glu 85 90 95 gga gac cct gtt ctt gct att ggt ggt cag gtt aag cgt gca gat ctc 336 Gly Asp Pro Val Leu Ala Ile Gly Gly Gln Val Lys Arg Ala Asp Leu 100 105 110 ttg aaa cgt gcc cac caa tca atg aat aac gtt gct atg ctt gag cca 384 Leu Lys Arg Ala His Gln Ser Met Asn Asn Val Ala Met Leu Glu Pro 115 120 125 att acc aaa tat gct gct gaa gta cat gat gct aac acc ctt tct gaa 432 Ile Thr Lys Tyr Ala Ala Glu Val His Asp Ala Asn Thr Leu Ser Glu 130 135 140 acg gtt gct aat gcc tat cgt cac gct aag tca ggg aaa cca ggt gca 480 Thr Val Ala Asn Ala Tyr Arg His Ala Lys Ser Gly Lys Pro Gly Ala 145 150 155 160 agc ttc att tca att cct caa gac gtg acg gat gct ccg gtc agt gtt 528 Ser Phe Ile Ser Ile Pro Gln Asp Val Thr Asp Ala Pro Val Ser Val 165 170 175 aag gct att aag cct atg aca gat cca aaa ctt ggt tca gca tct gtt 576 Lys Ala Ile Lys Pro Met Thr Asp Pro Lys Leu Gly Ser Ala Ser Val 180 185 190 tct gat att aac tat cta gca caa gcc att aaa aat gca gtg ttg cca 624 Ser Asp Ile Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro 195 200 205 gtc ttt ctt ttg ggg aat ggt gcc tca tca gaa gcc gta act tac tct 672 Val Phe Leu Leu Gly Asn Gly Ala Ser Ser Glu Ala Val Thr Tyr Ser 210 215 220 att cgc caa att ttg aag cat gtt aaa ttg cca gtt gtt gaa act ttc 720 Ile Arg Gln Ile Leu Lys His Val Lys Leu Pro Val Val Glu Thr Phe 225 230 235 240 caa ggt gcc ggt atc gtg tca cgt gac ctt gaa gaa gat act ttc ttt 768 Gln Gly Ala Gly Ile Val Ser Arg Asp Leu Glu Glu Asp Thr Phe Phe 245 250 255 ggt cgt gta ggt ctt ttc cgt aac caa ccc gga gac atg ttg ctt aaa 816 Gly Arg Val Gly Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys 260 265 270 aaa tcc gac tta gtt att gcc att ggt tat gat cca atc gaa tat gaa 864 Lys Ser Asp Leu Val Ile Ala Ile Gly Tyr Asp Pro Ile Glu Tyr Glu 275 280 285 gca cgt aac tgg aat gct gaa att tca gca cgt atc atc gtt att gat 912 Ala Arg Asn Trp Asn Ala Glu Ile Ser Ala Arg Ile Ile Val Ile Asp 290 295 300 gtc gag ccg gcc gag gtg gac act tac ttc caa ccg gaa cgt gaa ttg 960 Val Glu Pro Ala Glu Val Asp Thr Tyr Phe Gln Pro Glu Arg Glu Leu

305 310 315 320 att ggt aat gta gaa gcg agc tta gac ttg ctt ttg ccc gct att caa 1008 Ile Gly Asn Val Glu Ala Ser Leu Asp Leu Leu Leu Pro Ala Ile Gln 325 330 335 ggt tat aaa ttg cct gaa ggt gcg gtt gaa tat ctt aaa ggt ttg aaa 1056 Gly Tyr Lys Leu Pro Glu Gly Ala Val Glu Tyr Leu Lys Gly Leu Lys 340 345 350 aac aat gtt gtt gag gat gtt aag ttt gac cgt cag cct gat gaa ggt 1104 Asn Asn Val Val Glu Asp Val Lys Phe Asp Arg Gln Pro Asp Glu Gly 355 360 365 acg gtg cat ccg cta gat ttc atc gaa aat ttg caa gaa cac aca gat 1152 Thr Val His Pro Leu Asp Phe Ile Glu Asn Leu Gln Glu His Thr Asp 370 375 380 gat gat atg act gtt acg ttt gat gtt ggt agt cac tat att tgg atg 1200 Asp Asp Met Thr Val Thr Phe Asp Val Gly Ser His Tyr Ile Trp Met 385 390 395 400 gca cgt tat ctc aaa tcg tat gaa cca cgt cat ttg ctt ttc tca aat 1248 Ala Arg Tyr Leu Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn 405 410 415 ggg atg caa acg ata ggt att gct att aca tgg gct atc tct gca gca 1296 Gly Met Gln Thr Ile Gly Ile Ala Ile Thr Trp Ala Ile Ser Ala Ala 420 425 430 ttg gtt cgt cct aag aca aaa gtg att tct gta tct ggt gat ggt ggt 1344 Leu Val Arg Pro Lys Thr Lys Val Ile Ser Val Ser Gly Asp Gly Gly 435 440 445 ttc ctc ttc tca gca caa gaa ttg gaa aca gca gtt cgt ttg aaa ttg 1392 Phe Leu Phe Ser Ala Gln Glu Leu Glu Thr Ala Val Arg Leu Lys Leu 450 455 460 cca att gtc cat att atc tgg aac gat ggt cat tac aat atg gtg gaa 1440 Pro Ile Val His Ile Ile Trp Asn Asp Gly His Tyr Asn Met Val Glu 465 470 475 480 ttc cag gaa gaa atg aag tac ggt cgt tca tct ggg gtt gac ttt ggt 1488 Phe Gln Glu Glu Met Lys Tyr Gly Arg Ser Ser Gly Val Asp Phe Gly 485 490 495 cct gta gat ttt gta aaa tat gct gag agc ttt gga gcc aaa ggt tat 1536 Pro Val Asp Phe Val Lys Tyr Ala Glu Ser Phe Gly Ala Lys Gly Tyr 500 505 510 cgt gca aca agt aaa gca gcg ttt gct agc ttg ctt caa gag gct ttg 1584 Arg Ala Thr Ser Lys Ala Ala Phe Ala Ser Leu Leu Gln Glu Ala Leu 515 520 525 act cag gct gta gat gga cca gtc ctt att gat gtt cca att gac tat 1632 Thr Gln Ala Val Asp Gly Pro Val Leu Ile Asp Val Pro Ile Asp Tyr 530 535 540 aaa gat aac att aaa ctc ggc gaa act att ttg cca gat gaa ttt tac 1680 Lys Asp Asn Ile Lys Leu Gly Glu Thr Ile Leu Pro Asp Glu Phe Tyr 545 550 555 560 taa 1683 <210> SEQ ID NO 99 <211> LENGTH: 560 <212> TYPE: PRT <213> ORGANISM: Streptococcus thermophilus <400> SEQUENCE: 99 Val Phe Met Ser Glu Glu Lys Gln Leu Tyr Gly Ala Asp Leu Val Val 1 5 10 15 Asp Ser Leu Ile Asn His Asp Val Glu Tyr Val Phe Gly Ile Pro Gly 20 25 30 Ala Lys Ile Asp Arg Val Phe Asp Thr Leu Glu Asp Lys Gly Pro Glu 35 40 45 Leu Ile Val Ala Arg His Glu Gln Asn Ala Ala Phe Met Ala Gln Gly 50 55 60 Val Gly Arg Ile Thr Gly Lys Pro Gly Val Val Leu Val Thr Ser Gly 65 70 75 80 Pro Gly Val Ser Asn Leu Ala Thr Gly Leu Val Thr Ala Thr Asp Glu 85 90 95 Gly Asp Pro Val Leu Ala Ile Gly Gly Gln Val Lys Arg Ala Asp Leu 100 105 110 Leu Lys Arg Ala His Gln Ser Met Asn Asn Val Ala Met Leu Glu Pro 115 120 125 Ile Thr Lys Tyr Ala Ala Glu Val His Asp Ala Asn Thr Leu Ser Glu 130 135 140 Thr Val Ala Asn Ala Tyr Arg His Ala Lys Ser Gly Lys Pro Gly Ala 145 150 155 160 Ser Phe Ile Ser Ile Pro Gln Asp Val Thr Asp Ala Pro Val Ser Val 165 170 175 Lys Ala Ile Lys Pro Met Thr Asp Pro Lys Leu Gly Ser Ala Ser Val 180 185 190 Ser Asp Ile Asn Tyr Leu Ala Gln Ala Ile Lys Asn Ala Val Leu Pro 195 200 205 Val Phe Leu Leu Gly Asn Gly Ala Ser Ser Glu Ala Val Thr Tyr Ser 210 215 220 Ile Arg Gln Ile Leu Lys His Val Lys Leu Pro Val Val Glu Thr Phe 225 230 235 240 Gln Gly Ala Gly Ile Val Ser Arg Asp Leu Glu Glu Asp Thr Phe Phe 245 250 255 Gly Arg Val Gly Leu Phe Arg Asn Gln Pro Gly Asp Met Leu Leu Lys 260 265 270 Lys Ser Asp Leu Val Ile Ala Ile Gly Tyr Asp Pro Ile Glu Tyr Glu 275 280 285 Ala Arg Asn Trp Asn Ala Glu Ile Ser Ala Arg Ile Ile Val Ile Asp 290 295 300 Val Glu Pro Ala Glu Val Asp Thr Tyr Phe Gln Pro Glu Arg Glu Leu 305 310 315 320 Ile Gly Asn Val Glu Ala Ser Leu Asp Leu Leu Leu Pro Ala Ile Gln 325 330 335 Gly Tyr Lys Leu Pro Glu Gly Ala Val Glu Tyr Leu Lys Gly Leu Lys 340 345 350 Asn Asn Val Val Glu Asp Val Lys Phe Asp Arg Gln Pro Asp Glu Gly 355 360 365 Thr Val His Pro Leu Asp Phe Ile Glu Asn Leu Gln Glu His Thr Asp 370 375 380 Asp Asp Met Thr Val Thr Phe Asp Val Gly Ser His Tyr Ile Trp Met 385 390 395 400 Ala Arg Tyr Leu Lys Ser Tyr Glu Pro Arg His Leu Leu Phe Ser Asn 405 410 415 Gly Met Gln Thr Ile Gly Ile Ala Ile Thr Trp Ala Ile Ser Ala Ala 420 425 430 Leu Val Arg Pro Lys Thr Lys Val Ile Ser Val Ser Gly Asp Gly Gly 435 440 445 Phe Leu Phe Ser Ala Gln Glu Leu Glu Thr Ala Val Arg Leu Lys Leu 450 455 460 Pro Ile Val His Ile Ile Trp Asn Asp Gly His Tyr Asn Met Val Glu 465 470 475 480 Phe Gln Glu Glu Met Lys Tyr Gly Arg Ser Ser Gly Val Asp Phe Gly 485 490 495 Pro Val Asp Phe Val Lys Tyr Ala Glu Ser Phe Gly Ala Lys Gly Tyr 500 505 510 Arg Ala Thr Ser Lys Ala Ala Phe Ala Ser Leu Leu Gln Glu Ala Leu 515 520 525 Thr Gln Ala Val Asp Gly Pro Val Leu Ile Asp Val Pro Ile Asp Tyr 530 535 540 Lys Asp Asn Ile Lys Leu Gly Glu Thr Ile Leu Pro Asp Glu Phe Tyr 545 550 555 560 <210> SEQ ID NO 100 <211> LENGTH: 1665 <212> TYPE: DNA <213> ORGANISM: Vibrio angustum <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (1)..(1665) <400> SEQUENCE: 100 atg tcg gat aaa acc gtc tct ggt gct gaa ctg gtt gtt gaa act tta 48 Met Ser Asp Lys Thr Val Ser Gly Ala Glu Leu Val Val Glu Thr Leu 1 5 10 15 aat gca cat aac gtt cca cac att ttt ggt att cct gga gca aag gtg 96 Asn Ala His Asn Val Pro His Ile Phe Gly Ile Pro Gly Ala Lys Val 20 25 30 gat gct gtt ttc gat gct gtt tgt gat aac gga cca gaa atc att att 144 Asp Ala Val Phe Asp Ala Val Cys Asp Asn Gly Pro Glu Ile Ile Ile 35 40 45 tgt cat cat gaa caa aat gca gcg ttt atg gca gca gca act ggg cgt 192 Cys His His Glu Gln Asn Ala Ala Phe Met Ala Ala Ala Thr Gly Arg 50 55 60 tta acg ggt aaa gca ggc att tgt tta gca acc tct gga cca ggc gca 240 Leu Thr Gly Lys Ala Gly Ile Cys Leu Ala Thr Ser Gly Pro Gly Ala 65 70 75 80 tca aac ctt gtc aca ggc gtt gca aca gcg aat agt gaa ggt gat cct 288 Ser Asn Leu Val Thr Gly Val Ala Thr Ala Asn Ser Glu Gly Asp Pro 85 90 95 gtg gtt gca ctt gca ggt gct gta cct ctt tct atg tat tct cac aat 336 Val Val Ala Leu Ala Gly Ala Val Pro Leu Ser Met Tyr Ser His Asn 100 105 110 act cat caa tcc atg gat acc cgt tca ctg ttt act cct atc acc aag 384 Thr His Gln Ser Met Asp Thr Arg Ser Leu Phe Thr Pro Ile Thr Lys 115 120 125 ttt tca gca gaa gtg atg gat agc agc tcg gta tct gat gtt gta cat 432 Phe Ser Ala Glu Val Met Asp Ser Ser Ser Val Ser Asp Val Val His 130 135 140 aaa gct ttt cgt att gca gag caa cct acc caa ggt gct agc ttt gtt 480 Lys Ala Phe Arg Ile Ala Glu Gln Pro Thr Gln Gly Ala Ser Phe Val 145 150 155 160 agt cta ccg caa gat att cta act aac cgt att cct tac cag cca gta 528 Ser Leu Pro Gln Asp Ile Leu Thr Asn Arg Ile Pro Tyr Gln Pro Val 165 170 175 caa cag cct aat cca att ttg ttc ggt ggt gca cac cca caa gct att 576 Gln Gln Pro Asn Pro Ile Leu Phe Gly Gly Ala His Pro Gln Ala Ile 180 185 190 cgt cag gct gct gat cgc att aat gct gca aaa aat ccg gtg tta tta 624 Arg Gln Ala Ala Asp Arg Ile Asn Ala Ala Lys Asn Pro Val Leu Leu 195 200 205 ctg ggc atg gat gca agc cag cct ttt gtt gct gat gct att cgc caa 672 Leu Gly Met Asp Ala Ser Gln Pro Phe Val Ala Asp Ala Ile Arg Gln 210 215 220 cta ctc aaa caa aca cca att gcc gtt gtg aat acg ttt gcc gca gct 720 Leu Leu Lys Gln Thr Pro Ile Ala Val Val Asn Thr Phe Ala Ala Ala 225 230 235 240 ggg gtt att tct cat gat tta tac aac tgc ttt tta ggt cgt gtt ggc 768 Gly Val Ile Ser His Asp Leu Tyr Asn Cys Phe Leu Gly Arg Val Gly 245 250 255

tta ttt aaa aat caa ccc ggt gat att gca tta aac agt gca gat tta 816 Leu Phe Lys Asn Gln Pro Gly Asp Ile Ala Leu Asn Ser Ala Asp Leu 260 265 270 atc att acc att ggc tac agc cca att gaa tac gat ccg att ctt tgg 864 Ile Ile Thr Ile Gly Tyr Ser Pro Ile Glu Tyr Asp Pro Ile Leu Trp 275 280 285 aat aaa gat gca aac aca cca att att cat att ggt tat caa caa gca 912 Asn Lys Asp Ala Asn Thr Pro Ile Ile His Ile Gly Tyr Gln Gln Ala 290 295 300 gat tta gaa att agc tat aac cct gtt tgt gaa gtt gtg ggt gac tta 960 Asp Leu Glu Ile Ser Tyr Asn Pro Val Cys Glu Val Val Gly Asp Leu 305 310 315 320 gcg gtg tct gtc acg tct att gct tct gaa tta gat aag cga gaa tca 1008 Ala Val Ser Val Thr Ser Ile Ala Ser Glu Leu Asp Lys Arg Glu Ser 325 330 335 tta gaa aat aac caa caa atc caa tta tta cgc cac gat tta caa cat 1056 Leu Glu Asn Asn Gln Gln Ile Gln Leu Leu Arg His Asp Leu Gln His 340 345 350 att atg cag atg ggg gta aat aaa acc tca aca aac ggc gtt cac ccg 1104 Ile Met Gln Met Gly Val Asn Lys Thr Ser Thr Asn Gly Val His Pro 355 360 365 ctt cgt ttt gtt cat gag tta cgt cgc ttt gtt agt gac gac acc act 1152 Leu Arg Phe Val His Glu Leu Arg Arg Phe Val Ser Asp Asp Thr Thr 370 375 380 gta tgt tgt gat gta ggc tct att tat att tgg atg gca cgt tac ttc 1200 Val Cys Cys Asp Val Gly Ser Ile Tyr Ile Trp Met Ala Arg Tyr Phe 385 390 395 400 cac agc ttt gaa cct cgt cgt tta ttg ttc agc aat ggc caa caa aca 1248 His Ser Phe Glu Pro Arg Arg Leu Leu Phe Ser Asn Gly Gln Gln Thr 405 410 415 ttg ggc gta gct tta cct tgg gca att gca gct tcc ctt ctt cac cct 1296 Leu Gly Val Ala Leu Pro Trp Ala Ile Ala Ala Ser Leu Leu His Pro 420 425 430 aat gaa aaa gta att tcc atg tct ggt gat ggt ggc ttc cta ttc tca 1344 Asn Glu Lys Val Ile Ser Met Ser Gly Asp Gly Gly Phe Leu Phe Ser 435 440 445 tca atg gaa tta gcc acg gcc gtt cgc cat aaa tgt aat atc gtt cac 1392 Ser Met Glu Leu Ala Thr Ala Val Arg His Lys Cys Asn Ile Val His 450 455 460 ttt gtt tgg aca gat cac agt tat gac atg gtt aag atc caa cag ctt 1440 Phe Val Trp Thr Asp His Ser Tyr Asp Met Val Lys Ile Gln Gln Leu 465 470 475 480 aaa aag tat ggt cga gag agt gcc gtc agc ttt ata ggt cct gat att 1488 Lys Lys Tyr Gly Arg Glu Ser Ala Val Ser Phe Ile Gly Pro Asp Ile 485 490 495 gtt aag tac gca gaa agc ttc ggc gca cat ggt tta gcg atc aat act 1536 Val Lys Tyr Ala Glu Ser Phe Gly Ala His Gly Leu Ala Ile Asn Thr 500 505 510 gcc gat gat att gag cct gtt atg cga aaa gct atg agc tta agt ggc 1584 Ala Asp Asp Ile Glu Pro Val Met Arg Lys Ala Met Ser Leu Ser Gly 515 520 525 cca gta ttg gtc aac gtc aat gtt gat tat agc gat aac agt cgc cta 1632 Pro Val Leu Val Asn Val Asn Val Asp Tyr Ser Asp Asn Ser Arg Leu 530 535 540 ctt gat caa ctt cat cca tgc caa caa gat taa 1665 Leu Asp Gln Leu His Pro Cys Gln Gln Asp 545 550 <210> SEQ ID NO 101 <211> LENGTH: 554 <212> TYPE: PRT <213> ORGANISM: Vibrio angustum <400> SEQUENCE: 101 Met Ser Asp Lys Thr Val Ser Gly Ala Glu Leu Val Val Glu Thr Leu 1 5 10 15 Asn Ala His Asn Val Pro His Ile Phe Gly Ile Pro Gly Ala Lys Val 20 25 30 Asp Ala Val Phe Asp Ala Val Cys Asp Asn Gly Pro Glu Ile Ile Ile 35 40 45 Cys His His Glu Gln Asn Ala Ala Phe Met Ala Ala Ala Thr Gly Arg 50 55 60 Leu Thr Gly Lys Ala Gly Ile Cys Leu Ala Thr Ser Gly Pro Gly Ala 65 70 75 80 Ser Asn Leu Val Thr Gly Val Ala Thr Ala Asn Ser Glu Gly Asp Pro 85 90 95 Val Val Ala Leu Ala Gly Ala Val Pro Leu Ser Met Tyr Ser His Asn 100 105 110 Thr His Gln Ser Met Asp Thr Arg Ser Leu Phe Thr Pro Ile Thr Lys 115 120 125 Phe Ser Ala Glu Val Met Asp Ser Ser Ser Val Ser Asp Val Val His 130 135 140 Lys Ala Phe Arg Ile Ala Glu Gln Pro Thr Gln Gly Ala Ser Phe Val 145 150 155 160 Ser Leu Pro Gln Asp Ile Leu Thr Asn Arg Ile Pro Tyr Gln Pro Val 165 170 175 Gln Gln Pro Asn Pro Ile Leu Phe Gly Gly Ala His Pro Gln Ala Ile 180 185 190 Arg Gln Ala Ala Asp Arg Ile Asn Ala Ala Lys Asn Pro Val Leu Leu 195 200 205 Leu Gly Met Asp Ala Ser Gln Pro Phe Val Ala Asp Ala Ile Arg Gln 210 215 220 Leu Leu Lys Gln Thr Pro Ile Ala Val Val Asn Thr Phe Ala Ala Ala 225 230 235 240 Gly Val Ile Ser His Asp Leu Tyr Asn Cys Phe Leu Gly Arg Val Gly 245 250 255 Leu Phe Lys Asn Gln Pro Gly Asp Ile Ala Leu Asn Ser Ala Asp Leu 260 265 270 Ile Ile Thr Ile Gly Tyr Ser Pro Ile Glu Tyr Asp Pro Ile Leu Trp 275 280 285 Asn Lys Asp Ala Asn Thr Pro Ile Ile His Ile Gly Tyr Gln Gln Ala 290 295 300 Asp Leu Glu Ile Ser Tyr Asn Pro Val Cys Glu Val Val Gly Asp Leu 305 310 315 320 Ala Val Ser Val Thr Ser Ile Ala Ser Glu Leu Asp Lys Arg Glu Ser 325 330 335 Leu Glu Asn Asn Gln Gln Ile Gln Leu Leu Arg His Asp Leu Gln His 340 345 350 Ile Met Gln Met Gly Val Asn Lys Thr Ser Thr Asn Gly Val His Pro 355 360 365 Leu Arg Phe Val His Glu Leu Arg Arg Phe Val Ser Asp Asp Thr Thr 370 375 380 Val Cys Cys Asp Val Gly Ser Ile Tyr Ile Trp Met Ala Arg Tyr Phe 385 390 395 400 His Ser Phe Glu Pro Arg Arg Leu Leu Phe Ser Asn Gly Gln Gln Thr 405 410 415 Leu Gly Val Ala Leu Pro Trp Ala Ile Ala Ala Ser Leu Leu His Pro 420 425 430 Asn Glu Lys Val Ile Ser Met Ser Gly Asp Gly Gly Phe Leu Phe Ser 435 440 445 Ser Met Glu Leu Ala Thr Ala Val Arg His Lys Cys Asn Ile Val His 450 455 460 Phe Val Trp Thr Asp His Ser Tyr Asp Met Val Lys Ile Gln Gln Leu 465 470 475 480 Lys Lys Tyr Gly Arg Glu Ser Ala Val Ser Phe Ile Gly Pro Asp Ile 485 490 495 Val Lys Tyr Ala Glu Ser Phe Gly Ala His Gly Leu Ala Ile Asn Thr 500 505 510 Ala Asp Asp Ile Glu Pro Val Met Arg Lys Ala Met Ser Leu Ser Gly 515 520 525 Pro Val Leu Val Asn Val Asn Val Asp Tyr Ser Asp Asn Ser Arg Leu 530 535 540 Leu Asp Gln Leu His Pro Cys Gln Gln Asp 545 550 <210> SEQ ID NO 102 <211> LENGTH: 1689 <212> TYPE: DNA <213> ORGANISM: Bacillus cereus <400> SEQUENCE: 102 ttgagtacag gtgtaaaagc aaacgacgtg aagacaaaaa caaaaggagc agatcttgtt 60 gttgattgtt taattaaaca aggtgttaca catgttttcg gtattccagg agcaaagatt 120 gactctgtat ttgatgtact gcaagaaaga ggaccagagt taattgtttg tcgtcatgaa 180 caaaatgcag catttatggc agctgctatt ggtagattaa caggaaaacc gggcgtatgt 240 cttgtaactt caggaccagg gacatcaaat ttagcgacag gtcttgttac tgcgaatgcg 300 gagagtgatc ccgttgttgc tttagctggt gcagttccgc gtacggatcg attaaaacgt 360 acacatcaat ctatggataa tgctgcacta ttcgaaccaa tcacaaaata tagcgtagaa 420 gtagagcatc ctgataatgt gccagaagca ttatcaaatg cattccgaag tgcgacttct 480 acaaatccag gagcaacttt agtaagtttg ccgcaagacg ttatgactgc ggaaacgact 540 gtagagtcta tcggtgcgct ttctaagcca cagcttggaa tcgctcccac acatgatatt 600 acatatgtag tagataaaat aaaagcagcg aaattaccag ttattttact cggtatgaga 660 gcgagcacaa atgaagtgac gaaagccgtt cgtaaattaa ttgcggatac agaacttcct 720 gtcgttgaaa catatcaagc ggctggtgcc atttcacgtg agttagaaga tcatttcttc 780 ggccgtgttg gactattccg taaccaacca ggtgatattt tactagaaga ggcagatctt 840 gttatttcta tcggttatga tccaattgag tatgatccaa agttctggaa taaacttgga 900 gacagaacga ttattcatct tgatgaccat caagcagata tagatcatga ttaccaacca 960 gagcgtgaat taattggtga tattgcctta acagtaaata gcatcgcaga aaagttaccg 1020 aaacttgtgt taagtacgaa atcagaagca gtgttagaac gattacgcgc gaaattatca 1080 gaacaagcag aagttccaaa tcgtccttca gaaggtgtta cacatccgct tcaagtgatt 1140 cgtacacttc gttctttaat tagtgacgac acaaccgtta catgtgacat cggttcccat 1200 tctatttgga tggcgagatg tttccgttct tatgaaccac gtagattatt atttagtaac 1260 ggtatgcaga cgttaggtgt tgcacttcct tgggcaattg ctgctacttt agtagaacca 1320 ggtaaaaaag tagtttccgt atcaggtgac ggtggtttct tattctcagc gatggagtta 1380 gaaacggcgg tacgtttaaa ttctccaatc gtccatcttg tttggagaga cggcacatat 1440 gatatggttg cattccaaca aatgatgaaa tacggcagaa catcagctac agagtttggt 1500 gatgttgatc ttgttaaata tgcggaaagt ttcggggcgt taggtcttcg tgttaacacg 1560 cctgatgaat tagaaggggt attgaaagaa gcactagcag cagacggccc tgtcattatt 1620

gatattccaa ttgactatcg tgacaacatt aaattaagcg aaaaattatt accaaaccaa 1680 ttaaactaa 1689 <210> SEQ ID NO 103 <211> LENGTH: 562 <212> TYPE: PRT <213> ORGANISM: Bacillus cereus <400> SEQUENCE: 103 Met Ser Thr Gly Val Lys Ala Asn Asp Val Lys Thr Lys Thr Lys Gly 1 5 10 15 Ala Asp Leu Val Val Asp Cys Leu Ile Lys Gln Gly Val Thr His Val 20 25 30 Phe Gly Ile Pro Gly Ala Lys Ile Asp Ser Val Phe Asp Val Leu Gln 35 40 45 Glu Arg Gly Pro Glu Leu Ile Val Cys Arg His Glu Gln Asn Ala Ala 50 55 60 Phe Met Ala Ala Ala Ile Gly Arg Leu Thr Gly Lys Pro Gly Val Cys 65 70 75 80 Leu Val Thr Ser Gly Pro Gly Thr Ser Asn Leu Ala Thr Gly Leu Val 85 90 95 Thr Ala Asn Ala Glu Ser Asp Pro Val Val Ala Leu Ala Gly Ala Val 100 105 110 Pro Arg Thr Asp Arg Leu Lys Arg Thr His Gln Ser Met Asp Asn Ala 115 120 125 Ala Leu Phe Glu Pro Ile Thr Lys Tyr Ser Val Glu Val Glu His Pro 130 135 140 Asp Asn Val Pro Glu Ala Leu Ser Asn Ala Phe Arg Ser Ala Thr Ser 145 150 155 160 Thr Asn Pro Gly Ala Thr Leu Val Ser Leu Pro Gln Asp Val Met Thr 165 170 175 Ala Glu Thr Thr Val Glu Ser Ile Gly Ala Leu Ser Lys Pro Gln Leu 180 185 190 Gly Ile Ala Pro Thr His Asp Ile Thr Tyr Val Val Asp Lys Ile Lys 195 200 205 Ala Ala Lys Leu Pro Val Ile Leu Leu Gly Met Arg Ala Ser Thr Asn 210 215 220 Glu Val Thr Lys Ala Val Arg Lys Leu Ile Ala Asp Thr Glu Leu Pro 225 230 235 240 Val Val Glu Thr Tyr Gln Ala Ala Gly Ala Ile Ser Arg Glu Leu Glu 245 250 255 Asp His Phe Phe Gly Arg Val Gly Leu Phe Arg Asn Gln Pro Gly Asp 260 265 270 Ile Leu Leu Glu Glu Ala Asp Leu Val Ile Ser Ile Gly Tyr Asp Pro 275 280 285 Ile Glu Tyr Asp Pro Lys Phe Trp Asn Lys Leu Gly Asp Arg Thr Ile 290 295 300 Ile His Leu Asp Asp His Gln Ala Asp Ile Asp His Asp Tyr Gln Pro 305 310 315 320 Glu Arg Glu Leu Ile Gly Asp Ile Ala Leu Thr Val Asn Ser Ile Ala 325 330 335 Glu Lys Leu Pro Lys Leu Val Leu Ser Thr Lys Ser Glu Ala Val Leu 340 345 350 Glu Arg Leu Arg Ala Lys Leu Ser Glu Gln Ala Glu Val Pro Asn Arg 355 360 365 Pro Ser Glu Gly Val Thr His Pro Leu Gln Val Ile Arg Thr Leu Arg 370 375 380 Ser Leu Ile Ser Asp Asp Thr Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 400 Ser Ile Trp Met Ala Arg Cys Phe Arg Ser Tyr Glu Pro Arg Arg Leu 405 410 415 Leu Phe Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala 420 425 430 Ile Ala Ala Thr Leu Val Glu Pro Gly Lys Lys Val Val Ser Val Ser 435 440 445 Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala Val 450 455 460 Arg Leu Asn Ser Pro Ile Val His Leu Val Trp Arg Asp Gly Thr Tyr 465 470 475 480 Asp Met Val Ala Phe Gln Gln Met Met Lys Tyr Gly Arg Thr Ser Ala 485 490 495 Thr Glu Phe Gly Asp Val Asp Leu Val Lys Tyr Ala Glu Ser Phe Gly 500 505 510 Ala Leu Gly Leu Arg Val Asn Thr Pro Asp Glu Leu Glu Gly Val Leu 515 520 525 Lys Glu Ala Leu Ala Ala Asp Gly Pro Val Ile Ile Asp Ile Pro Ile 530 535 540 Asp Tyr Arg Asp Asn Ile Lys Leu Ser Glu Lys Leu Leu Pro Asn Gln 545 550 555 560 Leu Asn <210> SEQ ID NO 104 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 104 atctctcgag attacatcag aaaagacaac aa 32 <210> SEQ ID NO 105 <211> LENGTH: 35 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 105 cgatcccggg ttagtcatca ttttcatact gaatg 35 <210> SEQ ID NO 106 <211> LENGTH: 60 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 106 tcaaattatg gaggcgagaa acccgggatc gatggtacct aaatcggcat ttctagcatg 60 <210> SEQ ID NO 107 <211> LENGTH: 32 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 107 atcctgtaca actttgtaat acctgagtct ac 32 <210> SEQ ID NO 108 <211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 108 atagcccggg atataggagg aatttttgta atgttgacca aggctaccaa ag 52 <210> SEQ ID NO 109 <211> LENGTH: 33 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 109 tttaggtacc ttataacgct ttcgtcttca tta 33 <210> SEQ ID NO 110 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 110 ccaatgccta tctagctatg taag 24 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 111 agccttgttt caaccgatta 20 <210> SEQ ID NO 112 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 112 ttgttacttg attgcgactc g 21 <210> SEQ ID NO 113 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial sequence <220> FEATURE: <223> OTHER INFORMATION: Primer <400> SEQUENCE: 113 gatccaaatc aaaagcaact g 21

Claims

What is claimed is:

1. A recombinant lactic acid bacterial cell comprising at least one gene encoding a heterologous polypeptide having butanediol dehydrogenase activity and at least one genetic modification that reduces pyruvate formate lyase activity, wherein the bacterial cell is substantially free of lactate dehydrogenase activity, and wherein the cell comprises a 2-butanol or 2-butanone biosynthetic pathway.

2. The bacterial cell of claim 1 comprising a disruption in at least one endogenous gene encoding a polypeptide having lactate dehydrogenase activity.

3. The bacterial cell of claim 1 wherein the cell is a member of a genus selected from the group consisting of Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, Pediococcus, and Streptococcus.

4. The bacterial cell of claim 1 wherein the genetic modification affects a gene encoding pyruvare formate lyase, a gene encoding pyruvate formate lyase activating enzyme, or both.

5. The bacterial cell of claim 4 wherein the gene encoding pyruvate formate lyase is selected from the group consisting of pfl, pflB1 and pfl B2 and the gene encoding pyruvate formate lyase activating enzyme is selected from the group consisting of pflA, pflA1, and pflA2.

6. The bacterial cell of claim 2 wherein the polypeptide having lactate dehydrogenase activity is encoded by a gene selected from the group consisting of IdhL, IdhD, IdhL1, and IdhL2.

7. The bacterial cell of claim 6 herein the lactic acid host cell is Lactobacillus plantarum, Lactococcus lactis, Leuconostoc mesenteroides, Streptococcus thermophilus, Pediococcus pentosaceus, or Lactobacillus acidophilus.

8. The bacterial cell of claim 1 wherein the cell produces 2-butanone.

9. The bacterial cell of claim 1 wherein the cell produces 2-butanol.

10. The bacterial cell of claim 9 comprising a 2-butanol biosynthetic pathway, wherein the biosynthetic pathway comprises the following substrate to product conversions: a) pyruvate to acetolactate; b) acetolactate to acetoin; c) acetoin to 2,3-butanediol; d) 2,3-butanediol to 2-butanone; and e) 2-butanone to 2-butanol.

11. The bacterial cell of claim 8 comprising a 2-butanone biosynthetic pathway, wherein the biosynthetic pathway comprises the following substrate to product conversions: a) pyruvate to acetolactate; b) acetolactate to acetoin; c) acetoin to 2,3-butanediol; and d) 2,3-butanediol to 2-butanone.

12. A method for the production of 2-butanol comprising: a) providing the recombinant lactic acid bacterial cell of claim 1 comprising a 2-butanol biosynthetic pathway; and b) growing the bacterial cell of step (a) under conditions whereby 2-butanol is produced.

13. A method for the production of 2-butanone comprising: providing the recombinant lactic acid bacterial cell of claim 1 comprising a 2-butanone biosynthetic pathway; and b) growing the bacterial cell of step (a) under conditions whereby 2-butanone is produced.

14. The lactic acid bacterial cell of claim 1 comprising a 2-butanol biosynthetic pathway, wherein the heterologous polypeptide having butanediol dehydrogenase activity comprises an amino acid sequence with at least about 95% sequence identity to SEQ ID NO:13, SEQ ID NO: 64, or SEQ ID NO: 66.

15. The lactic acid bacterial cell of claim 14 wherein the heterologous polypeptide having butanediol dc: hydrogenase activity comprises the amino acid sequence of SEQ ID NO:13.

16. The lactic acid bacterial cell of claim 10 wherein the substrate to product conversion e) is catalyzed by a heterologous butanol dehydrogenase comprising an amino acid sequence with at least about 95% sequence identity to SEQ ID NO: 10.